Authors equally contributed to this work
The aim of our study was to determine the role of dystrophin hydrophobic regions in the pathogenesis of Duchenne (DMD) and Becker (BMD) muscular dystrophies, by the Kyte-Doolittle scale mean hydrophobicity profile and 3D molecular models. A total of 1038 cases diagnosed with DMD or BMD with the in-frame mutation were collected in our hospital and the Leiden DMD information database in the period 2002-2013. Correlation between clinical types and genotypes were determined on the basis of these two sources. In addition, the Kyte-Doolittle scale mean hydrophobicity of dystrophin was analyzed using BioEdit software and the models of the hydrophobic domains of dystrophin were constructed. The presence of four hydrophobic regions is confirmed. They include the calponin homology CH2 domain on the actin-binding domain (ABD), spectrin-type repeat 16, hinge III and the EF Hand domain. The severe symptoms of DMD usually develop as a result of the mutational disruption in the hydrophobic regions I, II and IV of dystrophin – those that bind associated proteins of the dystrophin-glycoprotein complex (DGC). On the other hand, when the hydrophobic region III is deleted, the connection of the ordered repeat domains of the central rod domain remains intact, resulting in the less severe clinical presentation. We conclude that mutational changes in the structure of hydrophobic regions of dystrophin play an important role in the pathogenesis of DMD.
Duchenne muscular dystrophy (DMD) is an X-linked, recessively inherited disease, typically characterized by a progressive skeletal muscle atrophy of proximate extremities and pseudohypertrophy of the gastrocnemius muscle [
Despite these differences in clinical severity, DMD and BMD are caused by a mutation of the same
For the large proteins, the 3D structure prediction remains a difficult and unresolved endeavor. Compared to ab initio prediction and fold recognition, a homology modeling - prediction based on the reasonable assumption that two homologous proteins will share very similar structures, is most accurate when the target and template have similar sequences. Dystrophin is a huge protein in humans, whose mapping is extremely challenging process. We used a Swiss model, a homology prediction method for remodeling parts of dystrophin. The results of the Swiss model are named
The aim of our study is to identify important functional domains of dystrophin by investigating the Kyte-Doolittle mean scale hydrophobicity profile [
A total of 1038 in-frame deletion mutation cases diagnosed with DMD or BMD were collected in the period from 2002 to 2013. 538 cases were collected in The First Affiliated Hospital of Sun Yat-sen University, while 500 cases were obtained from an open-access internet database - Leiden Muscular Dystrophy pages (
The MLPA test result was regarded as a genotype, while the clinical diagnosis (DMD/BMD) was considered as a phenotype. The genotypes and clinical phenotypes of 1038 in-frame deletion mutation cases were analyzed, and the numbers and percentages of cases that did not meet the reading-frame rule were calculated.
The amino acid sequence of
The three-dimensional structure of dystrophin was modeled by importing the dystrophin amino acid sequence to Swiss-model Automatic Modeling Mode [
The second structure information of 3D model and their corresponding functions were analyzed using RasMol software 2.7.2 (Biomolecular Structures Group, Glaxo Wellcome Research & Development, Stevenage, Hertfordshire, UK)[
SPSS V13.0 software (SPSS, Chicago, IL, USA) was employed to conduct statistical analyses. The two-sided chi-squared test was used to compare the difference between the percentages of genotypes or phenotype of DMD and BMD. The
Out of the 1038 analyzed in-frame dystrophin mutation cases, 242 (or 23.3%) showed DMD pattern, not meeting thus the criteria of the reading-frame rule. On the other hand, other 796 cases (76.7%) of the in-frame dystrophin mutations presented with BMD and were hence consistent with the reading-frame rule. These 1038 cases displayed over 93 distinct types of deletions (
The genotypes -phenotypes of 1038 cases with in-frame deletion The in-frame mutations of the 1038 analyzed patients include 93 genetically distinct mutations (horizontal lines). The numbers of cases of DMD/BMD for each of these distinct mutations are indicated on the right side of each horizontal line. 242 (23.3%) of 1038 in-frame mutation cases present symptoms of BMD, and hence meet the reading-frame rule. The number at the end of each horizontal bar is the number of cases with DMD versus BMD. The black and grey bars (from left to right) are the hydrophobic regions I-IV respectively.
The Kyte-Doolittle plot showed that dystrophin had four hydrophobic regions. These regions encompass residues 60-132 (forming greatest part of the actin-binding domain of dystrophin), 1990-2010 (forming part of the central rod domain), 2450-2500 (forming part of the central rod domain) and 3150-3300 forming the greatest part of the cysteine-rich domain, and were coded by exons 3-6, 42, 51, and 65-68, respectively. Their corresponding peak hydrophobicity values were: 0.3, 0.15, 0.34 and 0.29. The third hydrophobic region had the highest peak value (
Kyte & Doolittle Scale Mean Hydrophobicity Profile Analysis (A) Kyte & Doolittle Scale Mean Hydrophobicity Profile plot. Four hydrophobic regions are located at positions 60-132, 1990-2010, 2450-2500 and 3150-3300 of the dystrophin amino acid sequence (shown in the plot as marked with gray oval circles). Their corresponding peak values are 0.3, 0.15, 0.34 and 0.29. The third hydrophobic region has the highest peak value. (B) Involved hydrophobic regions and clinical type. A total of 715 cases (68.8%) have intact hydrophobic regions and there are 107/608 cases of DMD/BMD in this group. In contrast, when a hydrophobic region is involved in the mutation, the number of DMD/BMD cases is 135/188. The proportion of DMD (cases that do not meet the criteria of the reading-frame rule) in hydrophobic region-involved group (41.8%) is higher than the group in which hydrophobic regions are left intact (14.9%) (Χ2=88.09,
Hydrophobic regions were damaged by a gene deletion in 343 (33.0 %) of the 1038 in-frame mutations analyzed (
We have also examined contributions of each of the hydrophobic regions individually. As demonstrated in the
Homology models of the hydrophobic domains of dystrophin were built by using Swiss-model system. There are four 3D Batches including the whole or part of the hydrophobic regions. They are named as a Batch 1, a Batch 2, a Batch 3, and a Batch 4, respectively. These batches are separately included in the amino acid residues coded as 9-246, 2001-2307, 2471-2801, and 3047-3306, as demonstrated by the predicted 3D model. All of these 3D models were analyzed and displayed using RasMol software.
Batch 1 (
3D model of 4 hydrophobic regions-included amino acid residues (A) Batch 1 includes 2 dimers, which are formed by four dystrophin actin-binding domain (ABD) monomers. Each monomer is an extended dumbbell structure (panel E). Each dumbbell is composed of two calponin homology (CH) domains. Each monomer contains 3 actin-binding structures (ABS). Hydrophobic region I is located on the whole CH2 and the small beginning part of CH1 ABS3 (highlighted in green). (B, C) Batch 2 and 3 respectively. These batches are shown as three-stranded alpha-helical coiled coils. Hydrophobic region II contains (spectrin-type) repeat 16 of dystrophin (green on panel B). Hydrophobic region III includes 50 amino acid residues on the C-terminal part of hinge III (H III) and 30 amino acid residues on the N-terminal part of R20. H III is composed of irregular coils so that batch 3 only shows the 30 amino acids on the N-terminal part of R20 in a helical structure (highlighted in green). (D) Batch 4 is a globular structure that includes a hinge region, cysteine-rich domains on a WW domain and two helix-loop-helix EF hand-shaped structures. Hydrophobic region IV includes an EF hand-shaped region (shown in green). (E) The dystrophin actin-binding domain monomer is a dumbbell-shaped structure. Two calponin homology (CH) regions comprise the heads of the dumbbell. A triple helical bundle at the middle of monomer includes a helical linker (helix I). Seven helices form the N-terminal CH2 region (helical regions A-G). The CH1 region lacks the short B and D helices, which are replaced by a loop and an extra reverse turn on the N-terminal part of helix E. With the esception of the special highlighted parts, all the models were colored using RasMol software’s default coloring scheme for protein secondary structure: Alpha helices are colored in magenta, beta sheets are colored in yellow, turns are colored in pale blue, and all other residues are colored in white.
Batch 2 and Batch 3 (
Batch 4 (
Dystrophin is a membrane-bound cytoskeleton protein. It has been previously shown that it has four major function regions [
By using a scale mean hydrophobicity profile analysis method, we identified four hydrophobic regions in dystrophin. They are located between the residues 60 and 132, between the residues 1990 and 2010, between the residues 2450 and 2500 and between the residues 3150 and 3300 and coded by the exons 3-6, 42, 51, and 65-68, respectively.
Each monomer in the actin-binding domain is an extended dumbbell-shaped structure. The head of the dumbbell is formed by combining the two CH regions (CH1 and CH2). Hydrophobic region I includes the whole CH2 region and the small beginning part of the third actin-binding structures (ABS3) in the CH1 region. A previous investigation [
The second hydrophobic region is located in the central rod domain, which is formed by the linking of twenty-four spectrin-type repeats (STR) and four hinges [
The second hydrophobic region is located specifically on R16. Our study demonstrated that the involvement of this hydrophobic region in in-frame mutations broke the reading-frame rule and led to DMD in a relatively large percentage (76.9%) of patients. These mutations appear to impair the stability of the nNOS-sarcolemma connection, resulting in failure of nNOS to anchor onto the sarcolemma. In the study of Cazzella et al, the damage of nNOS-sarcolemma connection might be the cause of DMD [
The hydrophobicity of the third hydrophobic region is the strongest one. It is made up of residues 2450-2500, including 20 amino acid residues at the C-terminus of H III and 30 residues on the N-terminal part of R20. The peptide is coded by the exons 51 and 52, and includes an STR-separating hinge area rich in proline. Formation of alpha helices and beta sheets is more difficult when its sequence contains large number of proline. Therefore, this region may be predisposed to be disordered and flexible, and it is easy to extend and distort such a structure to some degree [
Our exploration of the fourth hydrophobic region informs us about the importance of dystrophin’s ability to bind β-dystroglycan. Dystrophin’s cysteine-rich region binds β-dystroglycan, and β-dystroglycan, in turn, is connected to the extracellular matrix [
In a summary, there are four hydrophobic regions in dystrophin, which are found on the CH2 portion of the ABD, in R16, in H III, and on an EF hand of cysteine-rich domain. These areas are important functional areas of dystrophin. Hydrophobic regions I, II and IV, respectively, affect the binding of actin, nNOS-sarcolemma, and β-dystroglycan, associated proteins of the DGC. Mutations of these hydrophobic regions thus directly impair formation of the DGC and consequent mutational damage, even when in-frame, results in the development of DMD phenotype. Hydrophobic region III contains the H III hinge and plays an important role in the stability of the dystrophin structure. The deletion of H III, in contrast, stretches the order of STR, causing less severe clinical symptoms than in patients with the intact H III. Taken together, the discovery of these hydrophobic regions and the understanding of their functionality in pathogenesis provide supplemental information to the reading-frame rule and support the strategies for the development of exon-skipping therapy [
The authors declare no conflict of interests.