A1型短指/趾症致病基因IHH的分子致病机理研究
摘要
A1型短指/趾症(Brachydactyly type A1, BDA1)是世界上第一例被记载的孟德尔常染色体显性遗传病(1903年),曾被许多遗传学和生物学教科书所引用,患者以中间指/趾骨变短、并可能与远端指/趾骨融合为主要特征,患者不仅伸手难看,而且影响正常生活与工作。到目前为止,全世界范围大概有超过100例的家系患者或者散在患者在医学杂志中被报道,但是在过去的一个世纪里,人们仍然没有找到导致A1型短指/趾的致病原因。在前期疾病基因定位克隆工作中,我们一共收集了三个A1型短指/趾大家系,将致病基因定位到了2号染色体长臂35到37的区域。最终发现位于这个区域、编码Indian hedgehog蛋白的IHH基因的突变导致了A1型短指/趾。我们在这三个不相关大家系的所有患者中分别确定了三个杂合错义突变,G283A(E95K), C300A(D100E)和G391A(E131K),它们均位于编码蛋白氨基端信号域的区域内。本研究在以上工作基础上,对IHH基因在体外功能方面进行了一系列的深入研究,并对其在A1型短指(趾)症致病机理方面的作用进行了探讨。
Ihh蛋白在翻译后早期是以一个全长的分子量约为46KD的前体蛋白形式存在,随后,在胆固醇脂的参与下,Ihh蛋白进行了自裂解,形成了一个20KD左右的C端有胆固醇修饰的IhhN信号活性蛋白和一个26KD左右的目前功能还不是很清楚的IhhC蛋白。信号活性蛋白IhhN在向细胞膜呈递过程中,又在N端进行了棕榈酸的修饰,在这两个脂修饰存在的情况下,并在dispatched蛋白的帮助下,IhhN蛋白以多聚体的形式从细胞膜脱离,向远端分泌到达靶细胞,并与其受体蛋白patched相结合,从而将信号进一步传递。本研究的重点就是探讨野生型和突变型Ihh蛋白在蛋白合成、加工、分泌运输以及与受体结合等方面的差别。
首先构建野生型和突变型IHH基因的真核表达载体,将这些载体瞬时转染ECHO细胞,然后用western-blotting检测细胞裂解液里目的蛋白水平。结果表明,三个突变体的IhhC蛋白产物的水平与野生型没有差别,但是E95K和D100E这两个突变体的信号活性蛋白IhhN部分明显比野生型减少很多,我们推测,三个突变体的分子内自裂解进程没有被破坏,但是可能E95K和D100E这两个突变体的IhhN蛋白的稳定性下降,容易被胞内蛋白酶所降解。另外,从它们的IhhC蛋白的表达水平没有差别来分析,这些突变蛋白的胆固醇修饰应该都是正常的,因为胆固醇依赖的自裂解反应都是正常的。为了了解三个突变型蛋白与野生型蛋白在向细胞膜呈递过程中是否存在差别,我们利用免疫荧光和显微共聚焦的方法对其进行了检测,结果发现,突变型蛋白都能够正确地呈递到细胞膜上,这说明突变蛋白的细胞膜呈递这一环节是正常的。接下来,我们通过检测转染细胞的条件性培养上清来分析突变型蛋白与野生型蛋白在从细胞膜向胞外分泌过程中是否存在差别,结果发现,E95K和E131K突变蛋白均与野生型蛋白一样能够正常分泌并在上清中检测到,但是,D100E突变蛋白却不能从条件性培养上清中检测到。我们知道,通常蛋白在不同温度情况下会表现出不同的生化特性,包括蛋白构像的变化和与其它分子相互作用的能力的改变等。在随后的试验中,我们探讨了突变蛋白和野生型蛋白在不同温度不同时间情况下的稳定性变化。试验结果表明,在37℃情况下,野生型蛋白与三个突变蛋白都存在不同程度的降解趋势,但是其中E95K和D100E这两个突变蛋白的降解速度更快,尤其是D100E蛋白,在表达3小时左右就几乎完全降解。而在30℃的情况下,所有的蛋白均能稳定存在。同时,在30℃情况下来检测D100E突变蛋白的细胞培养上清,发现能够检测到大量的D100E蛋白的存在,这表明,极有可能是因为在37℃情况下D100E突变蛋白以极快的速度被降解,从而导致在条件性上清中检测不到,而不是因为D100E蛋白不能被分泌。
从与IHH基因同源性极高的SHH (Sonic Hedgehog)基因蛋白的三维结构来分析,E95K和D100E这两个突变位点在空间位置上很接近,而且同属于一个位于蛋白分子表面的无规则卷曲结构域上,这个结构域由八个氨基酸(DEENTGAD)组成,根据突变点的位置,我们构建了几个截断体来验证这个区域对于整个Ihh蛋白的稳定性的影响。同时,从其他研究小组的报道中我们了解到还有两个突变也是位于这个区域的,有意思的是,这两个突变同样分别位于E95和D100的位置,不同的是,它们分别突变成了G和N,因此,我们同时也做了这两个突变体的表达分析。试验结果表明,这8个氨基酸的无规则卷曲区域对于IhhN蛋白的稳定性非常重要,如果将这八个氨基酸全部剔除,几乎检测不到IhhN蛋白的存在,而且,E95G和D100N这两个突变蛋白的表达情况与E95K和D100E这两个突变几乎相同,此结果提示这个区域内的几个突变所造成的BDA1的分子机制可能有相同之处。
硫酸肝素蛋白聚糖(HSPG)是广泛存在于细胞膜和细胞间质中的一类生物活性分子,有大量研究表明它在hedgehog信号通路中扮演重要角色。其中一个作用就是与hedgehog蛋白结合并协助其向远处运输。我们用亲和层析的方法来检测突变蛋白和野生型蛋白与heparin的结合能力上的差别,结果表明,E95K突变蛋白有着更强的与heparin结合的能力。这一结果提示E95K蛋白可能会在体内运输和组织分布过程中表现出与野生型不同的表型。
Hedgehog蛋白在组织间运输的过程还受到蛋白本身多聚体形式的影响,为此,我们利用分子筛层析的技术对突变型蛋白的多聚体形式进行了分析,结果发现E95K和E131K这两个突变蛋白的多聚体形成与野生型之间没有明显的差别。同时进行的棕榈酸修饰突变的蛋白没有多聚体形式存在,提示E95K和E131K这两个突变蛋白的棕榈酸修饰也是正常的。
Hedgehog蛋白与其受体Patched的结合是启动hedgehog信号传递的非常重要的一个环节,原核蛋白体外诱导C3H10T1/2细胞的结果表明三个突变型蛋白在诱导该细胞系向骨细胞方向分化的能力均比野生型蛋白有不同程度的下降,造成这一结果的最直接的可能原因就是突变型蛋白与受体Patched蛋白的结合能力下降。我们进行了野生型蛋白与突变型蛋白竞争结合受体Patched的试验,结果表明,三个突变型蛋白与受体Patched的结合能力都有不同程度地降低,其中以D100E降低最为严重,E95K突变蛋白和E131K突变蛋白分别降低2倍和1.5倍,它们的结合效应常数分别为:KI(E95K) = 40.6 nM;KI(E131K) = 30.5 nM;KI(D100E) >> 100 nM。
综上所述,本研究从Ihh蛋白的表达,加工修饰,分泌运输及受体结合等几个方面对三个突变蛋白的生化性质及体外功能与野生型蛋白进行了比较,发现了野生型与突变型蛋白之间的一些重要差异,并提出了突变型蛋白在体内异常传递运输的模型,这些新的发现为我们深入理解A1型短指(趾)症致病机理提供了重要线索,也为接下来的研究工作打下基础。
Brachydactyly type A-1 (BDA1) is the first recorded disorder of the autosomal dominant Mendelian trait (1903), which is cited in many genetic and biological textbooks. BDA1 is characterized by pronounced shortening or missing of the middle phalanges. Over 100 familial or sporadic cases from different ethnic groups have so far been reported. However, the real breakthrough in identifying the cause of BDA1 has not taken place until very late last century and early this century, when the Bio-X Center, Shanghai Jiao Tong University and Chinese Academy of Sciences, mapped and cloned the causative gene. We recruited three large families of BDA1 in the previous studies and mapped the locus for BDA1 on chromosome 2q35-q37 where we discovered mutations in Indian Hedgehog (IHH) was the cause for BDA1. In total we identified three heterozygous missense mutations (G283A (E95K), C300A (D100E) and G391A (E131K)) in the region encoding the amino-terminal signaling domain in all affected members of all three large, unrelated families. The three mutant amino acids, which are conserved across all vertebrates and invertebrates studied so far, are predicted to be adjacent on the surface of Ihh. In this work, we attempted to study the functional character of the mutants in order to understand the molecular basis of BDA1.
Ihh protein is a secreted morphogen that is essential for endochondral bone development. Ihh is synthesized as a 46 kDa precursor that undergoes autocatalytic cleavage into an active 19 kDa N-terminal fragment (IhhN) which is subsequently modified by attachment of cholesterol and palmitic acid, and a more divergent C-terminal domain (IhhC). The long-range effects of Ihh during embryonic development are facilitated by heparan sulfate proteoglycans (HSPGs) in the surrounding intracellular matrix and a soluble multimeric form. The IhhN product triggers Hh pathway activation by binding to Patched (Ptc), the primary receptor for Hh signaling. The emphasis of this study is to explore the difference of the synthesis, processing, secretion and receptor binding between wild type and mutant Ihh.
The eukaryotic constructs of wild type and mutant IHH gene were generated and transfected into ECHO cell line. Consequently, western-blotting analysis for the cell lysates indicated that the intermolecular cleavage and the cholesterol modification of three mutant Ihh were all normal and the IhhN fragment of E95K and D100E was unstable and degraded easily. In addition, the cell membrane location were all normal for three mutant proteins by immunofluorescence and confocal microscope. However, the assay of conditional culture medium implicated that the secretion of E95K and E131K mutants were normal but not D100E. Furthermore, temperature sensitivity analysis suggested that D100E protein was more unstable than E95K in 37℃and all proteins displayed the same stability in 30℃. Anyway, the secretion of D100E was normal in 30℃. Taken together, we speculated that D100E could't secrete because of its degradation in short time.
3D structure of ShhN protein indicated that the position of E95K and D100E mutations is in an eight amino acid (8aa) conserved domain. Subsequently, we generated some truncation constructs and western-blotting analysis indicated that the 8aa domain is very important for Ihh protein because the IhhN fragment of T6 and T8 was degraded easily. Interestingly, we found that the IhhN fragment was unstable for E95G and D100N like E95K and D100E mutants and this implicated the mechanism of these two mutants associated with BDA1 may be consistent with E95K and D100E.
Affinity chromatography assay implicated that the binding potency between E95K protein and heparin was enhanced and it is possible to result in the variety of the protein diffusion pattern. Gel chromatography assay indicated that the multimeric form of E95K and E131K were consistent with wild type. In addition, we suggested that the palmitoylation of E95K and E131K mutant proteins were normal because it could't form multimer for Hedgehog protein without palmitoylation.
The IhhN product triggers Hh pathway activation by binding to Ptc receptor. The induction assay in C3H10T1/2 cell line by Prokaryotic IhhN proteins implicated that the ability of three mutant proteins to induce Hedgehog signaling was significantly impaired compared with wild type, suggesting these mutants may affect interaction with the receptor Ptc either directly or indirectly. Subsequently, the binding assay indicated that the interaction potency of three mutant proteins to Ptc were significantly reduced compared with wild type, their KI value were listed below: E95K = 40.6nM; E131K = 30.5nM; D100E >> 100nM, whereas KD of wild type was 20.6nM.
Taken together, the present study analyzed the functional character including protein expression, modification, secretion and receptor binding of three mutants compared with wild type Ihh, and found some differences among them. Thereby, we could understand well the molecular mechanism of BDA1 from these findings.
Ihh蛋白在翻译后早期是以一个全长的分子量约为46KD的前体蛋白形式存在,随后,在胆固醇脂的参与下,Ihh蛋白进行了自裂解,形成了一个20KD左右的C端有胆固醇修饰的IhhN信号活性蛋白和一个26KD左右的目前功能还不是很清楚的IhhC蛋白。信号活性蛋白IhhN在向细胞膜呈递过程中,又在N端进行了棕榈酸的修饰,在这两个脂修饰存在的情况下,并在dispatched蛋白的帮助下,IhhN蛋白以多聚体的形式从细胞膜脱离,向远端分泌到达靶细胞,并与其受体蛋白patched相结合,从而将信号进一步传递。本研究的重点就是探讨野生型和突变型Ihh蛋白在蛋白合成、加工、分泌运输以及与受体结合等方面的差别。
首先构建野生型和突变型IHH基因的真核表达载体,将这些载体瞬时转染ECHO细胞,然后用western-blotting检测细胞裂解液里目的蛋白水平。结果表明,三个突变体的IhhC蛋白产物的水平与野生型没有差别,但是E95K和D100E这两个突变体的信号活性蛋白IhhN部分明显比野生型减少很多,我们推测,三个突变体的分子内自裂解进程没有被破坏,但是可能E95K和D100E这两个突变体的IhhN蛋白的稳定性下降,容易被胞内蛋白酶所降解。另外,从它们的IhhC蛋白的表达水平没有差别来分析,这些突变蛋白的胆固醇修饰应该都是正常的,因为胆固醇依赖的自裂解反应都是正常的。为了了解三个突变型蛋白与野生型蛋白在向细胞膜呈递过程中是否存在差别,我们利用免疫荧光和显微共聚焦的方法对其进行了检测,结果发现,突变型蛋白都能够正确地呈递到细胞膜上,这说明突变蛋白的细胞膜呈递这一环节是正常的。接下来,我们通过检测转染细胞的条件性培养上清来分析突变型蛋白与野生型蛋白在从细胞膜向胞外分泌过程中是否存在差别,结果发现,E95K和E131K突变蛋白均与野生型蛋白一样能够正常分泌并在上清中检测到,但是,D100E突变蛋白却不能从条件性培养上清中检测到。我们知道,通常蛋白在不同温度情况下会表现出不同的生化特性,包括蛋白构像的变化和与其它分子相互作用的能力的改变等。在随后的试验中,我们探讨了突变蛋白和野生型蛋白在不同温度不同时间情况下的稳定性变化。试验结果表明,在37℃情况下,野生型蛋白与三个突变蛋白都存在不同程度的降解趋势,但是其中E95K和D100E这两个突变蛋白的降解速度更快,尤其是D100E蛋白,在表达3小时左右就几乎完全降解。而在30℃的情况下,所有的蛋白均能稳定存在。同时,在30℃情况下来检测D100E突变蛋白的细胞培养上清,发现能够检测到大量的D100E蛋白的存在,这表明,极有可能是因为在37℃情况下D100E突变蛋白以极快的速度被降解,从而导致在条件性上清中检测不到,而不是因为D100E蛋白不能被分泌。
从与IHH基因同源性极高的SHH (Sonic Hedgehog)基因蛋白的三维结构来分析,E95K和D100E这两个突变位点在空间位置上很接近,而且同属于一个位于蛋白分子表面的无规则卷曲结构域上,这个结构域由八个氨基酸(DEENTGAD)组成,根据突变点的位置,我们构建了几个截断体来验证这个区域对于整个Ihh蛋白的稳定性的影响。同时,从其他研究小组的报道中我们了解到还有两个突变也是位于这个区域的,有意思的是,这两个突变同样分别位于E95和D100的位置,不同的是,它们分别突变成了G和N,因此,我们同时也做了这两个突变体的表达分析。试验结果表明,这8个氨基酸的无规则卷曲区域对于IhhN蛋白的稳定性非常重要,如果将这八个氨基酸全部剔除,几乎检测不到IhhN蛋白的存在,而且,E95G和D100N这两个突变蛋白的表达情况与E95K和D100E这两个突变几乎相同,此结果提示这个区域内的几个突变所造成的BDA1的分子机制可能有相同之处。
硫酸肝素蛋白聚糖(HSPG)是广泛存在于细胞膜和细胞间质中的一类生物活性分子,有大量研究表明它在hedgehog信号通路中扮演重要角色。其中一个作用就是与hedgehog蛋白结合并协助其向远处运输。我们用亲和层析的方法来检测突变蛋白和野生型蛋白与heparin的结合能力上的差别,结果表明,E95K突变蛋白有着更强的与heparin结合的能力。这一结果提示E95K蛋白可能会在体内运输和组织分布过程中表现出与野生型不同的表型。
Hedgehog蛋白在组织间运输的过程还受到蛋白本身多聚体形式的影响,为此,我们利用分子筛层析的技术对突变型蛋白的多聚体形式进行了分析,结果发现E95K和E131K这两个突变蛋白的多聚体形成与野生型之间没有明显的差别。同时进行的棕榈酸修饰突变的蛋白没有多聚体形式存在,提示E95K和E131K这两个突变蛋白的棕榈酸修饰也是正常的。
Hedgehog蛋白与其受体Patched的结合是启动hedgehog信号传递的非常重要的一个环节,原核蛋白体外诱导C3H10T1/2细胞的结果表明三个突变型蛋白在诱导该细胞系向骨细胞方向分化的能力均比野生型蛋白有不同程度的下降,造成这一结果的最直接的可能原因就是突变型蛋白与受体Patched蛋白的结合能力下降。我们进行了野生型蛋白与突变型蛋白竞争结合受体Patched的试验,结果表明,三个突变型蛋白与受体Patched的结合能力都有不同程度地降低,其中以D100E降低最为严重,E95K突变蛋白和E131K突变蛋白分别降低2倍和1.5倍,它们的结合效应常数分别为:KI(E95K) = 40.6 nM;KI(E131K) = 30.5 nM;KI(D100E) >> 100 nM。
综上所述,本研究从Ihh蛋白的表达,加工修饰,分泌运输及受体结合等几个方面对三个突变蛋白的生化性质及体外功能与野生型蛋白进行了比较,发现了野生型与突变型蛋白之间的一些重要差异,并提出了突变型蛋白在体内异常传递运输的模型,这些新的发现为我们深入理解A1型短指(趾)症致病机理提供了重要线索,也为接下来的研究工作打下基础。
Brachydactyly type A-1 (BDA1) is the first recorded disorder of the autosomal dominant Mendelian trait (1903), which is cited in many genetic and biological textbooks. BDA1 is characterized by pronounced shortening or missing of the middle phalanges. Over 100 familial or sporadic cases from different ethnic groups have so far been reported. However, the real breakthrough in identifying the cause of BDA1 has not taken place until very late last century and early this century, when the Bio-X Center, Shanghai Jiao Tong University and Chinese Academy of Sciences, mapped and cloned the causative gene. We recruited three large families of BDA1 in the previous studies and mapped the locus for BDA1 on chromosome 2q35-q37 where we discovered mutations in Indian Hedgehog (IHH) was the cause for BDA1. In total we identified three heterozygous missense mutations (G283A (E95K), C300A (D100E) and G391A (E131K)) in the region encoding the amino-terminal signaling domain in all affected members of all three large, unrelated families. The three mutant amino acids, which are conserved across all vertebrates and invertebrates studied so far, are predicted to be adjacent on the surface of Ihh. In this work, we attempted to study the functional character of the mutants in order to understand the molecular basis of BDA1.
Ihh protein is a secreted morphogen that is essential for endochondral bone development. Ihh is synthesized as a 46 kDa precursor that undergoes autocatalytic cleavage into an active 19 kDa N-terminal fragment (IhhN) which is subsequently modified by attachment of cholesterol and palmitic acid, and a more divergent C-terminal domain (IhhC). The long-range effects of Ihh during embryonic development are facilitated by heparan sulfate proteoglycans (HSPGs) in the surrounding intracellular matrix and a soluble multimeric form. The IhhN product triggers Hh pathway activation by binding to Patched (Ptc), the primary receptor for Hh signaling. The emphasis of this study is to explore the difference of the synthesis, processing, secretion and receptor binding between wild type and mutant Ihh.
The eukaryotic constructs of wild type and mutant IHH gene were generated and transfected into ECHO cell line. Consequently, western-blotting analysis for the cell lysates indicated that the intermolecular cleavage and the cholesterol modification of three mutant Ihh were all normal and the IhhN fragment of E95K and D100E was unstable and degraded easily. In addition, the cell membrane location were all normal for three mutant proteins by immunofluorescence and confocal microscope. However, the assay of conditional culture medium implicated that the secretion of E95K and E131K mutants were normal but not D100E. Furthermore, temperature sensitivity analysis suggested that D100E protein was more unstable than E95K in 37℃and all proteins displayed the same stability in 30℃. Anyway, the secretion of D100E was normal in 30℃. Taken together, we speculated that D100E could't secrete because of its degradation in short time.
3D structure of ShhN protein indicated that the position of E95K and D100E mutations is in an eight amino acid (8aa) conserved domain. Subsequently, we generated some truncation constructs and western-blotting analysis indicated that the 8aa domain is very important for Ihh protein because the IhhN fragment of T6 and T8 was degraded easily. Interestingly, we found that the IhhN fragment was unstable for E95G and D100N like E95K and D100E mutants and this implicated the mechanism of these two mutants associated with BDA1 may be consistent with E95K and D100E.
Affinity chromatography assay implicated that the binding potency between E95K protein and heparin was enhanced and it is possible to result in the variety of the protein diffusion pattern. Gel chromatography assay indicated that the multimeric form of E95K and E131K were consistent with wild type. In addition, we suggested that the palmitoylation of E95K and E131K mutant proteins were normal because it could't form multimer for Hedgehog protein without palmitoylation.
The IhhN product triggers Hh pathway activation by binding to Ptc receptor. The induction assay in C3H10T1/2 cell line by Prokaryotic IhhN proteins implicated that the ability of three mutant proteins to induce Hedgehog signaling was significantly impaired compared with wild type, suggesting these mutants may affect interaction with the receptor Ptc either directly or indirectly. Subsequently, the binding assay indicated that the interaction potency of three mutant proteins to Ptc were significantly reduced compared with wild type, their KI value were listed below: E95K = 40.6nM; E131K = 30.5nM; D100E >> 100nM, whereas KD of wild type was 20.6nM.
Taken together, the present study analyzed the functional character including protein expression, modification, secretion and receptor binding of three mutants compared with wild type Ihh, and found some differences among them. Thereby, we could understand well the molecular mechanism of BDA1 from these findings.
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