计算机三维重构验证Dystrophin疏水区段与Duchenne型肌营养不良的发病

doi:10.3969/j.issn.2095-4344.2013.50.014

中国组织工程研究

• 组织构建基础实验 basic experiments in tissue construction • 上一篇下一篇

计算机三维重构验证Dystrophin疏水区段与Duchenne型肌营养不良的发病

梁颖茵¹，操基清¹，杨娟²，张成¹

1中山大学附属第一医院，广东省广州市 510000；2南方医科大学珠江医院神经内科，广东省广州市 510000

修回日期:2013-11-15 出版日期:2013-12-10 发布日期:2013-12-10
通讯作者: 张成，博士，博士生导师，教授，中山大学附属第一医院神经内科，广东省广州市 510700
作者简介:梁颖茵☆，中山大学在读博士，主治医师，主要从事神经遗传及肌肉病方面的研究。共同第一作者：操基清，中山大学在读博士，主要从事神经内科方面的研究。共同第一作者：杨娟，主要从事神经内科方面的研究。

Effects of the dystrophin hydrophobic regions in the pathogenesis of Duchenne muscular dystrophy
A three-dimensional reconstruction verification

Liang Ying-yin¹, Cao Ji-qing¹, Yang Juan², Zhang Cheng¹

1 The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510000, Guangdong Province, China
2 Department of Neurology, Zhujiang Hospital of Southern Medical University, Guangzhou 510000, Guangdong Province, China

Revised:2013-11-15 Online:2013-12-10 Published:2013-12-10
Contact: Zhang Cheng, M.D., Doctoral supervisor, Professor, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510000, Guangdong Province, China chengzhang100@hotmail.com
About author:Liang Ying-yin☆, Studying for doctorate, Attending physician, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510000, Guangdong Province, China liangyingyin@hotmail.com Cao Ji-qing, Studying for doctorate, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510000, Guangdong Province, China Yang Juan, Department of Neurology, Zhujiang Hospital of Southern Medical University, Guangzhou 510000, Guangdong Province, China

摘要/Abstract

摘要：

背景：Duchenne型肌营养不良和Becker型进行性肌营养不良都是dystrophin基因突变所致，但后者临床表型较轻。“阅读框规则”可解释大部分基因型与临床型关系，但累及疏水区段的整码突变也可导致Duchenne型肌营养不良。因此很有必要了解疏水区域在dystrophin中的功能，且这些疏水区段的三维结构及功能在发病机制中所起的具体作用仍未阐明。
目的：通过Kyte&Doolittle平均疏水轮廓分析研究dystrophin的疏水区段。利用swiss-model三维重构dystrophin的疏水区段阐述其在发病机制中所起的作用。
方法：参考莱顿开放数据库(http://www.dmd.nl/)及收集中山大学附属第一医院2002年至2013年确诊Duchenne型进行性肌营养不良或Becker型进行性肌营养不良的缺失型整码突变患者资料共1 038例，分析其临床型与基因型关系。使用bioedit软件计算dystrophin的平均疏水轮廓及利用swiss-model三维重构疏水区段，结合临床型和基因型关系确定dystrophin重要功能区。
结果与结论：dystrophin存在4个疏水区段，分别为肌动蛋白结合区内的Calponin同源区2、中央棒区内的重复区16、第三铰链区和EF手型区。第1，2，4疏水区段是dystrophin糖蛋白复合物中dystrophin与其他糖蛋白的结合区域，其破坏严重影响dystrophin糖蛋白复合物功能，临床症状重。中央棒区在第三铰链区附近断裂后，HⅢ的无规则卷结构不容易与断端重复区的螺旋结构恢复连接。但第三铰链区同时缺失，其两端的重复区较容易重新连接，所以第3疏水区破坏后其临床症状反而较轻。提示dystrophin的疏水区段是其重要功能区，多是dystrophin糖蛋白复合物中dystrophin与相关蛋白的结合部位，在Duchenne型肌营养不良的发病机制中起重要作用。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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关键词: 组织构建, 组织构建基础实验, Duchenne型肌营养不良, Becker型进行性肌营养不良, dystrophin, Kyte&Doolittle疏水轮廓分析, 三维结构, bioedit, swiss-model, 基因型-临床型分析

Abstract:

BACKGROUND: Duchenne muscular dystrophy is recognized as a fatal X-linked recessive inheritance. It is caused by the dystrophin gene mutation, resulting in the deficiency of dystrophin and consequent degeneration and necrosis of muscle fibers gradually. Becker muscular dystrophy is also caused by the mutation of the same gene, but presented with less severe clinical symptoms compared with Duchenne muscular dystrophy. Frameshift mutation destroys the reading frames, and thus the translation cannot proceed smoothly to transcript functional proteins. In-frame mutation cannot destroy the reading frames and hence the translation can proceed smoothly. But in-frame mutation involves the whole hydrophobic regions. The three-dimensional structure of these regions and their functionality are not interpreted clearly. The effects of these regions on disease development need to be clarified in detail from the point of structure and function.
OBJECTIVE: By analyzing Kate and Dolittle scale mean hydrophobicity profile, to investigate the dystrophin hydrophobic regions using Swiss-model so as to provide the supplement explanation on the reading frame rule.
METHODS: Form 2002 to 2013, 1 038 cases diagnosed as Duchenne muscular dystrophy or Becker muscular dystrophy were collected in the First Hospital of Sun Yat-sen University in China and Leiden DMD information database was searched with deletion of codon mutation information available. The correlation between clinical types and genotypes was analyzed upon resources collected above. The mean hydrophobicity profile of dystrophin was analyzed by Bioedit as well as the reconstruction of hydrophobic domains using Swiss-model. Thus, the important functional domain of dystrophin was confirmed by analysis and the correlation between clinical types and genotypes.
RESULTS AND CONCLUSION: Four hydrophobic regions were confirmed: Calponin homology domain CH2 on actin-binding domain, repeat 16 domain, Hinge Ⅲ domain and EF Hand domain. Duchenne muscular dystrophy was developed as a result of the destruction of the 1st, 2nd and 4th hydrophobic regions which were the conjunction of dystrophin and associated protein in dystrophin-glycoprotein complex. When the 3rd hydrophobic was deleted, the repeat domain located on central rob domain remained its continuity so that the clinical symptoms were less severe. These findings indicate that the dystrophin hydrophobic regions act as an important role on the pathogenesis of Duchenne muscular dystrophy.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: muscular dystrophy, Duchenne, hydrophobic and hydrophilic interactions, protein structure, tertiary, protein conformation

中图分类号:

R318

梁颖茵，操基清，杨娟，张成. 计算机三维重构验证Dystrophin疏水区段与Duchenne型肌营养不良的发病[J]. 中国组织工程研究, doi: 10.3969/j.issn.2095-4344.2013.50.014.

Liang Ying-yin, Cao Ji-qing, Yang Juan, Zhang Cheng. Effects of the dystrophin hydrophobic regions in the pathogenesis of Duchenne muscular dystrophy
A three-dimensional reconstruction verification[J]. Chinese Journal of Tissue Engineering Research, doi: 10.3969/j.issn.2095-4344.2013.50.014.

图/表（结果） 4

The genotypes-phenotypes of 1 038 cases of in-frame deletion
796 of 1 038 cases met the criteria of the reading-frame rule, accounting for 76.7%. In the rest 242 cases (23.3%), 102 types of deletion forms were identified (Table 1).

Kyte & Doolittle Scale Mean Hydrophobicity Profile Analysis
Kyte & Doolittle curve showed there were four hydrophobic regions on dystrophin, relatively located on 60-132, 1 990-2 010, 2 450-2 500 and 3 150-3 300th amino acid sequences. They were coded by 3-6, 42, 51, 65-68 exons, respectively. Their corresponding peak values were 0.3, 0.15, 0.34 and 0.29. The 3rd hydrophobic region owned the highest peak value (Figure 4).

Hydrophobic regions and phenotypes
In all 1 038 cases, 714 cases (68.8%) had integrated hydrophobic regions; the rest 324 cases (31.2%) belonged to the deletion forms (Table 2). In the group with integrated hydrophobic regions, there were 104 cases of DMD (14.6%) and 610 cases of BMD (85.4%).

In the deletion form group, there were 137 cases of DMD (42.3%) and 187 of BMD (57.7%). The proportion of DMD in two groups (cases who did not met the criteria of the reading frame rule) had a significantly statistical difference (P < 0.001). As seen from Table 2, BMD was more common in the integrated hydrophobic region groups and the 3rd region involvement alone than DMD. In other involved hydrophobic regions, DMD incidence was higher than that of BMD.

3D Swiss-modeling for the dystrophin hydrophobic regions By using Swiss-model, a homology structure was forecasted on the important domains of dystrophin. Four hydrophobic regions were separately included in the amino acid residues coded as 9-246, 2 001-2 307, 2 471-2 801, 3 047-3 306, as demonstrated by the 3D predicted model. They were named as Batches 1-4 respectively. All the 3D-models were showed and analyzed in Rasmol software (Figure 5).

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引言

Duchenne muscular dystrophy (DMD) is an X-linked recessive inherited disease with the typical clinical performance of progressive skeletal muscle atrophy of proximate extremities and the pseudohypertrophy in gastrocnemius muscle. The patients usually have its onset between the age of 3-5 and lose their walking ability at the age of 13-15 and most die around age of 20 owning to respiratory failure or/and cardiac insufficiency^[1-3]. The cause of DMD is mainly due to the gene mutation in X chromosome which results in lack of dystrophin locating on muscular cell membrane. Becker muscular dystrophy (BMD) is similar to DMD in terms of clinical performance, but with a slighter severity. BMD patients can maintain their
walking ability even after 15. Both DMD and BMD are due to the mutation of the same gene sequence but have obvious differences on the severity of disease. The former is considered as a fatal genetic disorder while the latter is of longer life span and better life quality^[4-7]. DMD and BMD are caused by the gene mutation of dystrophin which is a cytoskeleton protein and its gene is located on Xp21.1^[8]. Dystrophin is a large cytoskeleton protein with molecular mass of 427 kDa. It is composed of N-terminal region, central rob domain, C-terminal region. The N-terminal region of dystrophin binds the actin. The large central rob domain comprises 24 spectrin-like repeats and 4 hinges, and another actin binding side and a nNOS binding side are found in central rob dmain. The C-terminal region binds the dystrobrevins, syntrophins and β-dystroglycan, a transmembrane glycoprotein, which have interaction with sarcoglycans and α-dystroglycan. Furthermore, α-dystroglycan has interaction with laminin-2. That is to say, the membrane-spanning dystrophin glycoprotein complex (DGC) act as an indirect linkage between the actin-based cytoskeleton and the extracellular matrix^[9-10]. DMD gene is the largest gene in human being. There are 79 exons in it and various mutation types are found in the gene, 65% are deletion, 7% to 10% are duplication, 25% to 30% are point mutation^[11-14]. Out-of-frame mutation can interrupt the open reading frame (ORF) so that the transcription is terminated. Therefore the dystrophin production is not sufficient to elicit the function. In-frame mutation did not destroy ORF and thus dystrophin is shortened only and its partial function remains. General speaking out-of-frame mutation causes DMD while in-frame mutation results in BMD; this is termed as “reading-frame rule”^[15-16]. However, some in-frame mutations can also cause DMD^[17], which is hard to explain by this rule. Also, this kind of situation indicates the sequence location where has mutation will have important influences on the structure and function of in-frame mutation^[18]. This aim of study is by investigating the Kate & Doolittle mean scale hydrophobicity profile^[19-20], to indentify the important functional domains of dystrophin and to constitute its structure in a three-dimensional (3D) model. Their structure and functions will be analyzed as well as the pathogenesis mechanism when they are destroyed. By this way, supplemental information is provided to explain the reading-frame rule.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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材料方法

Design

A retrospective analysis.

Time and setting

This study was performed at the First Affiliated Hospital of Sun Yat-Sen University, China from October 2012 to February 2013.

Materials

Case resources

Form 2002 to 2013, 1 038 cases diagnosed as DMD/ BMD were collected in the First Hospital of Sun Yat-sen University, and Leiden Muscular Dystrophy pages^[21-22] (http://www.dmd.nl) were searched with deletion of in-frame mutation information available. Assessment of mutation types was performed using a Reading-frame Checker (DMD Exonic Deletions/Duplications Reading- Frame Checker 1.9, http://www.humgen.nl/scripts/ DMD_frame).

Documented multiplex probe ligation dependent amplification (MLPA) testing results are available to confirm the gene mutation type.

Diagnosis of DMD and BMD clinical types^[23]

DMD is defined if patients cannot walk before 12 years of age and those have significant clinical symptoms and signs meeting the diagnosis criteria of DMD even below 12 years of age. BMD patients can usually still walk at 16. In some patients present with the clinical symptoms and signs which meet the criteria of BMD, they are even younger than 16 years of age. Intermediate muscular dystrophy (IMD) patients are aged between 12-16 years of age. For those subjects younger than 12 years of age and non-significant signs presented to differentiate types, DMD/BMD is the preferred term to diagnosis.

Inclusion criteria: (1) Definite diagnosis of DMD or BMD. (2) Available DNA diagnosis result with MLPA.

Exclusion criteria: (1) IMD. (2) Due to age not meet diagnosis criteria

Methods

Genotype-phenotype analysis

The genotypes and clinical types of 1 038 in-frame deletion mutation cases were analyzed; the numbers and proportions of cases which did not meet the rule were calculated by excel software.

Kate & Doolittle scale mean hydrophobicity profile analysis

The amino acid sequence of dystrophin (NCBI Reference Sequence: NP_003997.1) from gene bank (http://www.ncbi.nlm.nih.gov/genbank) was traced and imported into Bioedit software 7.0.1(Tom Hall Ibis Biosciences, An Abbott company, Carlsbad, CA)^[24] to conduct Kate & Doolittle scale mean hydrophobicity profile analysis. By setting the window side at 56^[25], the four hydrophobic peak values were calculated separately as shown on Figure 1. We compared the involvement of hydrophobic regions with their relationship with clinical types and calculated the proportion of DMD in the integrated hydrophobic regions and deletion types in groups.

Remodel dystrophin 3D structure

The 3D structure of dystrophin was remodeled by importing the dystrophin amino acid sequence to Swiss- model Automatic Modelling Mode^[26] (http://swissmodel. expays.org). This modeling system can turn amino acid sequences into 3D homology structure automatically. 3D models can be downloaded from the myWorkspace on Swiss-model web server.

3D model analysis by Rasmol software

The 2nd tier structure of 3D model and their corresponding functions were demonstrated by Rasmol software 2.7.2 (Biomolecular Structures Group, Glaxo Wellcome Research & Development, Stevenage, Hertfordshire, UK)^[27]. 3D models’ demonstration display and color allocation were setup by Structure and Ribbons respectively (Figure 2). Then we checked the second structure information in Rasmol command line window (Figure 2) and manually chosed the hydrophobic regions color in green(Figure 3).

Main outcome measures

The proportion of in-frame deletion cases meeting the reading-frame rule; the effects of dystrophin hydrophobic regions on phenotypes.

Statistical analysis

SPSS 13.0 software was used to conduct statistical analysis. Chi-square test was used to compare the rates

with a significant level at 0.05 at two-side.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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讨论

Dystrophin is a membrane-bound cytoskeleton protein. It has been know that there are four major function regions^[32]: actin-binding domain (ABD), central rob domain, cysteine-rich domain and carboxyl terminal. Dystrophin connects to other glucoproteins to from a complex to DGC. By this way, the complex can stabilize the cell membrane and transit the signal cross membrane^[33]. The stability of protein conformation and interaction between proteins is heavily relied on the intramolecular hydrophobic effects^[34]. The contents of hydrophobic amino acid in the hydrophobic regions in dystrophin are richer than other regions. Its impairment will be mostly leading to DMD, even in the presence of in-frame mutation, suggesting the importance of hydrophobic region in dystrophin. We tried to use Bioinformatics techniques to remodel these hydrophobic regions and analyzed their function.

By using scale mean hydrophobicity profile analysis method, we identified that in dystrophin, four hydrophobic regions are existing. They are located on 60-132, 1 990-2 010, 2 450-2 500 and 3 150-3 300^th amino acid sequences, respectively, and coded by exon 3-6, 42, 51, 65-68 respectively.

First hydrophobic region includes the whole CH2 region and the small beginning part of ABS3 in CH1 region. Previous investigation^[33] found that actin is directly combined with ABS1 and ABS3 in ABD. ABS1 and ABS3 are located on helix A of CH1 and CH2 respectively. ABS2 is located on helix G and F in CH1 region. They do not stay on the same planum. Conformation change will be happened in CH region when binding to actin protein leading to the arrangement of ABC. A junction surface is finally formed as a result. CH2 region works as an assistance region to increase the affinity of connection between ABD and actin^[35]. In this study, 59.2% of patients with in-frame mutation did not meet the rule and have clinical symptoms of DMD. This may be due to the serious damage of CH2, directly affecting the connection affinity between ABD and actin. However the conformation change on the junction surface did not perform a significant change. This can be explained in some BMD cases. Twenty-four spectrin-type-repeats and four Hinges are linked together to form dystrophin central rob domain. Each STR is composed by 110 amino acid residues ^[36]. HingeⅠ(HⅠ) connects the amino terminal of dystrophin and N terminal of central rob domain. HingeⅡ(HⅡ) connects R3 and R4; Hing Ⅲ(HⅢ) links R19 and R20; Hinge Ⅳ(HⅣ) is the connection between C terminal of central rob domain and N terminal of cysteine-rich domain^[37-39]. Batch 2 and 3 of the 3D reconstruction in this study is located on the central rob domain, all shown as a Coliform structure with three helix peptides overlapped. They contain 52 helixes with 76 turns and 11 helixes with 22 turns, as well as some irregular scrolls. No β overlap is identified. Batch2 is where R16 to R18 are located. It is considered as a combination area with neuronal nitric oxide synthase (nNOS)^[40-41^]. R16/17 is the minimum unit to anchor on sacrolemma. R16/17 α2 and α3 helixes are the only and necessary to the anchoring of nNOS-sacrolemma connection, which requires the α2 and α3 helixes on R16 and 3 helixes on R17. R17 α1 helix links to nNOS directly via the connection micro junction region. Other helixes can stabilize the catenating structure. nNOS-sarcolemma is involved into the post-injury recovery of muscle activity and signal transduction.

Second hydrophobic region is located on R16. Our study demonstrated that the involvement of this region led to DMD in 76.9% of patients with in-frame mutation. Stability of nNOS-sarcolemma is impaired, as a result of this region involvement. As a consequent, nNOS fail to anchor on sarcolemma, followed by cell death^[42]. The hydrophobicity of the 3^rd hydrophobic is strongest. It is made up of 2 450-2 500 amino acids, including 50 amino acids at HⅢ C terminal and 30 amino acids on R20 N terminal. The peptide is coded by 51 and 52 exons. Hinge area is homology region to separate spectrin-type-repeat, enriching with proline. It is hard to form helix and plate sheet structure. Therefore disordered structure is predisposed to generate and good flexibility is available. This kind of structure is easy to develop extension and distortion in some degree^[43-44]. Current investigations show that the order of STR elicits a significant influences on clinical symptoms^[45]. If HⅢ does exist, its irregular coil structure is no so easy to re-conjunct with the helix structure of repeat domain. In contrast, HⅢ deletion will make spectrin-type-repeat at both ends connected to repeat domain again, thus restoring the STR sequence. The clinical symptoms of this group of patients have less severe than those with STR conjunction errors. It is also supported by our data that 79.6% patients with the involvement of 3^rd hydrophobic region are BMD. In our study, unlike the traditional hypothesis^[46], which considered HⅢ is an important part to maintain the stability of dystrophin and its deletion will cause DMD, the deletion of HⅢ has various influences on DGC function, subject to individual performance. One study focusing on the stability, structure and lipid binding properties of products after exon 51 skipping-therapy indicate that the products after therapy are various in patients^[47]. In another study with

targeted on exon 51 skipping in MDX mice, similar phenomenon is also investigated^[48]. Therefore the role of HⅢ in DGC still needs further investigation. Dystrophin is in conjunction with β-dystroglycan via cysteine-rich region. β-dystroglycan is connected to the extracellular matrix^{[49-50]-3 092^nd amino acids. When β-dystroglycan combines with dystrophin, WW domain is embedded into neighboring EF hand structure to become a bigger complex^[51-52]. Proline-rich region of β-dystroglycan directly conjuncts EF hand structure which also stabilizes WW domain. The 4^th hydrophobic peptide is located in EF hand structure and coded by 65-66 exons. Its damage directly influences the connection point between dystrophin and β-dystroglycan. As a result, DGC function is seriously affected and DMD occurs.}. Cysteine-rich region contains WW domain, EF hand 1, EF hand 2 and ZZ domain. By computerized 3D reconstruction, Batch4 is shown as a ball structure, including two EF hand structure. Thirty-one α helixes and 31 β helixes forms into this batch via 40 reverse turn. Most of them are dystrophin cysteine-rich domain WW and EF hand structure. WW domain is composed of 3 056

In a summary, there are four hydrophobic regions in dystrophin, including CH2 on ABD, R16, HⅢ, EF hand. These areas are important functional areas of dystrophin. 1^st, 2^nd and 4^th hydrophobic regions are the conjunction part between dystrophin and associated proteins in DGC. The involvement of 1^st, 2^nd and 4^th hydrophobic regions will respectively affect the conjunction of dystrophin and actin, nNOS-sarcolemma, dystrophin and β-dystroglycan. DGC is impaired in a direct way. DMD is resulted from the damage to these regions even in the presence of in-frame mutation. 3^rd hydrophobic peptide is where HⅢ exists, which plays an important role in the stability of dystrophin structure. The deletion of HⅢ, in contrast, stretches the order of spectrin-type-repeat. The clinical symptoms of this group of patients have less severe than those with integrated HⅢ. This may be related to individual differences of the role of HⅢ in DGC. Discovery of these hydrophobic regions and the understanding to their functionality provide the supplement information to the reading rule from the pathogenesis and support to build up the strategy of exon skipping therapy^[53-54].

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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计算机三维重构验证Dystrophin疏水区段与Duchenne型肌营养不良的发病

Effects of the dystrophin hydrophobic regions in the pathogenesis of Duchenne muscular dystrophy
A three-dimensional reconstruction verification

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计算机三维重构验证Dystrophin疏水区段与Duchenne型肌营养不良的发病

Effects of the dystrophin hydrophobic regions in the pathogenesis of Duchenne muscular dystrophy A three-dimensional reconstruction verification

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Effects of the dystrophin hydrophobic regions in the pathogenesis of Duchenne muscular dystrophy
A three-dimensional reconstruction verification