Abstract
Masticatory muscle tendon–aponeurosis hyperplasia (MMTAH) is a new disease associated with limited mouth opening that is often misdiagnosed as a temporomandibular disorder; subsequently, patients are mistakenly treated with irreversible operations. Due to the poor presentation and characterization of symptoms, the underlying pathological conditions remain unclear. We have previously conducted a proteomic analysis of tendons derived from one MMTAH subject and one facial deformity subject using two-dimensional fluorescence difference gel electrophoresis and liquid chromatography coupled with tandem mass spectrometry. However, the results were obtained for only one subject. The aim of the present study was to confirm the expression of specific molecules in tendon tissues from multiple subjects with MMTAH by applying two-dimensional polyacrylamide gel electrophoresis with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Of the 19 proteins identified in tendons from both MMTAH and facial deformity patients, fibrinogen fragment D and beta-crystallin A4 were up-regulated, whereas myosin light chain 4 was down-regulated in MMTAH. We also found fibrinogen to be expressed robustly in tendon tissues of MMTAH patients. Our data provide the possibility that the distinctive expression of these novel proteins is associated with the pathology of MMTAH.
Masticatory muscle tendon–aponeurosis hyperplasia (MMTAH) is a newly described disease associated with limited mouth opening. The condition advances very slowly from adolescence; both mandibular angles exhibit hyperplasia, resulting in a characteristic square mandible. MMTAH is diagnosed by a palpable dense band at the anterior border of the masseter muscle upon maximum mouth opening and a square mandible configuration. Hyperplasia of tendons and aponeuroses in MMTAH leads to a disturbance in muscle extensibility, resulting in limited mouth opening. Common useful diagnostic markers of this disease are currently unavailable because of its poor objective symptoms.
Although MMTAH was formally approved as a disease at the conference of The Japanese Society of the Temporomandibular Joint in 2008, it is not well recognized in other countries. A lack of recognition of this disease among general practitioners results in misdiagnosis of the temporomandibular disorder, and patients have often been treated with irreversible operations. Therefore, there is a need to establish criteria for the correct diagnosis of MMTAH as soon as possible. From the viewpoint of histopathology, Chiba observed that the tendons and aponeuroses in MMTAH appear normal because of a lack of both inflammation and transformation; he therefore suggested that the excess tissue is a result of hyperplasia. We have previously demonstrated that a good long-term recovery is reliably obtained in patients with MMTAH via resection of the hyperplastic masseter muscle aponeuroses and coronoidectomy for separation of the temporal muscle from the coronoid process.
Tendons are structurally composed of tenocytes lying within a network of extracellular matrix. They are the tissues that connect the muscle to the bone and transmit forces generated by the muscle to the bone. Although proteomic analysis has been performed on murine tendons, a lack of studies in man leaves it unclear as to which proteins are expressed in human tendons. Facial deformity (FD), a disease that is defined as a congenital or acquired condition of the skeleton in the head and face region, requires surgical correction for the deformities. In contrast to MMTAH patients, FD patients have normal tendons that do not exhibit hyperplasia.
Tendon tissues in MMTAH appear normal upon microscopic observation and gross pathology. However, the aberrant sound heard upon cutting with scissors indicates degenerative changes at the electron microscope or protein level. Furthermore, MMTAH progresses bilaterally and is a juvenile-onset disease. We hypothesize that disease progression involves both environmental and genetic factors. We have previously conducted a preliminary study to identify candidate molecules as potential diagnostic markers in MMTAH using a two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) system and liquid chromatography coupled with tandem mass spectrometry. However, that study involved only one test subject and one control subject, and used crude tendons that contained muscle tissues.
The aim of the present study was to confirm the expression of specific molecules in tendon tissues from multiple subjects with MMTAH by applying another approach. We thus performed proteomic analysis of tendons from three test subjects and three control subjects using a 2D-polyacrylamide gel electrophoresis (2D-PAGE) system and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Materials and methods
Subjects
Tissue specimens from the temporal tendons of four subjects with MMTAH and four with FD whose tendon tissue showed no sign of hyperplasia and who had no limited mouth opening, were obtained from patients undergoing surgery ( Table 1 ). None of the patients had underlying diseases. The dentist in charge provided all the patients or their guardians with an explanation of the study. The participants provided their written informed consent to participate in this study. Patients were free to withdraw from the study at any time. Informed consent was obtained from all subjects. Three samples from the MMATH group and three from the FD group (i.e., M1, M2, M3, F1, F2, and F3) were subjected to proteome analysis, and one sample from each group (i.e., M4 and F4) was subjected to histological analysis.
| Diagnosis | No. | Sex | Age, years |
|---|---|---|---|
| MMTAH | M1 | Female | 35 |
| MMTAH | M2 | Female | 41 |
| MMTAH | M3 | Female | 44 |
| MMTAH | M4 | Female | 42 |
| FD | F1 | Female | 25 |
| FD | F2 | Male | 29 |
| FD | F3 | Male | 19 |
| FD | F4 | Male | 41 |
Sample preparation
Each tendon tissue (150–200 mg wet weight of tissue) with the muscle completely removed under a stereomicroscope, was homogenized using an SK mill (Tokken Inc., Japan) under liquid nitrogen cooling, and then lysed in a buffer containing 7 M urea, 2 M thiourea, 30 mM Tris (tris(hydroxymethyl)aminomethane), 3% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and 1% Triton X-100. The resultant mixture was sonicated for 20 s. The homogenate was then centrifuged at 40,000 rpm for 60 min at 20 °C; the supernatant (0.5–3 ml) was subsequently dialyzed using a Slide-A-Lyzer Dialysis Cassettes kit at 3.5 K molecular weight cut-off (Thermo Scientific Inc., USA) overnight and then dried in a SpeedVac vacuum centrifuge (Thermo Scientific). The sample was the lysed again in the same buffer. Protein concentrations were determined using a Pierce 660 nm Protein Assay (Thermo Scientific).
2D gel electrophoresis
2D gel electrophoresis was performed as described previously. Briefly, the sample was mixed in a rehydration solution containing 8 M urea, 2% CHAPS, 2% immobilized pH gradient (IPG) buffer, 0.3% dithiothreitol (DTT), and a few grains of bromophenol blue dye, and the resulting mixture was vortexed. The first dimension was performed with an Ettan IPGphor II (GE Healthcare, USA) using IPG strips pH 3–10 L, 18 cm (GE Healthcare). The second dimension was performed with a Multiphor II 2-D (GE Healthcare) using ExcelGel SDS XL Gradient 12–14%, 245 cm × 180 cm (GE Healthcare). For the first dimension, an aliquot of sample supernatant containing 1.0 mg protein was loaded onto an IPG strip, and the strip was then allowed to rehydrate overnight. The first dimension was performed with the following voltage programme: 100 V for 12 h (step-and-hold), 500 V for 1 h (gradient), 1000 V for 1 h (gradient), 8000 V for 1 h (gradient), and 8000 V for 40 h (step-and-hold). Prior to the second-dimension separation, IPG strips were incubated for 20 min in an equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS), and bromophenol blue) containing 0.2% DTT, followed by equilibration in the same buffer containing 4% iodoacetamide and bromophenol blue dye instead of DTT. The IPG strip was then placed cathode side down on top of the SDS gel, and second-dimension electrophoresis was performed under the following conditions: 1000 V, 20 mA, and 40 W for 45 min; 1000 V, 40 mA, and 40 W for 5 min; and 1000 V, 40 mA, and 40 W for 160 min.
Protein spot image analysis
The separated protein spots were visualized using Coomassie brilliant blue (CBB) G-250, and the 2D gel electrophoresis gels were digitized using a GS-800 Calibrated Densitometer (Bio-Rad, USA). Scanned data were entered into Discovery Series PDQuest 2D Gel Analysis software (Ver. 7.10; Bio-Rad).
In-gel digestion, mass spectrometry, and database analysis
Protein spots were excised and then processed via in-gel digestion using the following protocol. The gel piece was immersed in 100 μl of 50% methanol and placed on an ultrasonic device for 10 min. Any liquid was then discarded. This step was repeated four times. Next, the gel piece was immersed in 100 μl of 50% acetonitrile for 10 min, and then in 100 μl of 100% acetonitrile for 10 min. The gel piece was subsequently immersed in 100 μl of 50 mM ammonium bicarbonate minimum for 10 min, and then in 100 μl of 100% acetonitrile for 10 min. Following a second immersion in 100 μl of 50 mM ammonium bicarbonate for at least 10 min, the gel piece was then placed in 100 μl of 50% acetonitrile/50 mM ammonium bicarbonate for at least 10 min. Finally, there was an immersion in 100 μl of 100% acetonitrile for 10 min, and this step was repeated two times. The gel piece was then dried by removing all liquid, and then placed in 5 μl of trypsin (Promega, USA) containing 20 μg/50 mM ammonium bicarbonate. The container was sealed using an adhesive seal to prevent evaporation, and the gel piece was incubated overnight in the digestion reagent at 37 °C. The next day, the gel piece was incubated in 15 μl of 45% acetonitrile/0.1% trifluoroacetic acid (TFA) on an ultrasonic device for 10 min, and the liquid was subsequently collected. This step was repeated twice. Finally, the extracts were dried in a SpeedVac vacuum centrifuge for 20 min. Dried peptides were then mixed with 5 μl of 0.1% TFA and an aliquot of 1 μl was placed on a target plate for MALDI-TOF MS with 1 μl of matrix solution (high quality alpha-cyano-4-hydroxycinnamic acid (CHCA); Shimadzu, Japan) and then analyzed using MALDI-TOF MS (Shimadzu, Japan). The resulting spectra were acquired in the delayed extraction and reflector mode under standard conditions (20 kV acceleration voltage, 120 ns delay time). The mass scale was calibrated internally with CHCA, [M 1 H]1 = 190.05, and adrenocorticotropic hormone (ACTH), [M 1 H]1 = 2465.20. Typically, 256 scans were averaged to produce the final spectrum. Peptide mass fingerprint (PMF) data, together with isoelectric point (pI) and molecular mass (Mr) values (estimated from 2D gel electrophoresis), were used to search the Matrix Science protein database using the programme MASCOT ( http://www.matrixscience.com/search_form_select.html ).
Densitometric analysis
Semi-quantification of identified spots was performed by densitometry with the Image J programme. We examined all samples of FD and MMTAH.
Histological analysis
For histological analysis, fixed tissues were embedded in paraffin and sectioned in 5-μm slices. Sections were processed for standard haematoxylin and eosin (H&E) staining or immunohistochemical procedures using anti-human fibrinogen (DAKO, Denmark) as a primary antibody, and observed under an upright microscope.
Statistical analysis
Comparisons between the two groups were analyzed using the Student’s t -test. In all analyses, a two-tailed probability of less than 5% ( P < 0.05) was considered to be statistically significant.
Results
To determine which proteins were up- or down-regulated in MMTAH in comparison to FD, we performed a 2D-PAGE analysis. A total of 29 expressed spots were excised from gels and subjected to protein analysis by MALDI-TOF MS. As a result of the PMF analysis, 24 spots of interest were successfully obtained ( Tables 2 and 3 ).
| Spot ID | Theoretical isoelectric point | Theoretical molecular weight | Sequence coverage (%) | Approved gene name |
|---|---|---|---|---|
| 1 | 5.85 | 109.709 | 6 | Collagen alpha-2(VI) chain |
| 2 | 6.97 | 79,310 | 23 | Serotransferrin precursor |
| 3 | 5.92 | 67,773 | 16 | PRO2619 |
| 4 | 5.37 | 44,280 | 16 | Chain A, alpha 1-antitrypsin |
| 5 | 5.84 | 38,081 | 22 | Fibrinogen beta chain |
| 6 | 5.19 | 37,125 | 20 | Actin peptide |
| 7 | 5.84 | 38,081 | 23 | Chain B, crystal structure of fibrinogen fragment D |
| 8 | 4.66 | 32,945 | 18 | Tropomyosin beta chain isoform 1 |
| 9 | 5.23 | 42,366 | 18 | Actin, alpha skeletal muscle |
| 10 | 5.23 | 42,334 | 14 | Actin, alpha cardiac muscle 1 proprotein |
| 11 | 5.84 | 38,081 | 14 | Chain B, crystal structure of fibrinogen fragment D |
| 12 | 4.97 | 21,189 | 33 | Myosin light chain 1/3, skeletal muscle isoform 1f |
| 13 | 5.27 | 28,061 | 26 | Apolipoprotein A-1 |
| 14 | 5.84 | 38,081 | 18 | Chain B, crystal structure of fibrinogen fragment D |
| 15 | 4.92 | 18,777 | 27 | Myosin regulatory light chain 2, ventricular/cardiac muscle isoform |
| 16 | 4.92 | 18,777 | 27 | Myosin regulatory light chain 2, ventricular/cardiac muscle isoform |
| 17 | 4.89 | 19,102 | 33 | Myosin regulatory light chain 2, skeletal muscle isoform |
| 18 | 5.66 | 18,486 | 23 | Thiol-specific antioxidant enzyme |
| 19 | 5.83 | 22,645 | 29 | Beta-crystallin A4 |
| 20 | 5.50 | 13,809 | 58 | Transthyretin |
| 21 | 5.34 | 15,048 | 33 | Galectin-1 |
| 22 | 6.03 | 11,073 | 66 | Haemoglobin beta chain |
| 23 | 6.03 | 11,073 | 63 | Haemoglobin beta chain |
| Spot ID | Theoretical isoelectric point | Theoretical molecular weight | Sequence coverage (%) | Approved gene name |
|---|---|---|---|---|
| 1 | 5.85 | 109.709 | 11 | Collagen, type VI, alpha 2 |
| 2 | 6.81 | 79,280 | 14 | PRO1400 |
| 3 | 5.92 | 70,564 | 17 | Albumin, isoform CRA_h |
| 4 | 5.51 | 46,978 | 21 | Alpha 1-antitrypsin |
| 5 | 5.84 | 38,081 | 18 | Chain B, crystal structure of fibrinogen fragment D |
| 6 | 5.39 | 38,142 | 16 | Actin, alpha skeletal muscle-like isoform3 |
| 7 | 5.84 | 38,081 | 15 | Chain B, crystal structure of fibrinogen fragment D |
| 8 | 4.66 | 32,945 | 22 | Tropomyosin beta chain isoform 1 |
| 9 | 5.26 | 32,370 | 20 | Actin, alpha 1, skeletal muscle |
| 10 | 4.88 | 30,498 | 14 | Actin, alpha, cardiac muscle, isoform CRA_c |
| 11 | 5.84 | 38,081 | 14 | Chain B, crystal structure of fibrinogen fragment D |
| 12 | 4.97 | 21,189 | 33 | Myosin light chain 1/3, skeletal muscle isoform |
| 13 | 5.27 | 28,061 | 24 | Apolipoprotein A-1 |
| 15 | 4.92 | 18,777 | 32 | Myosin regulatory light chain 2, ventricular/cardiac muscle isoform |
| 16 | 4.92 | 18,777 | 32 | Myosin regulatory light chain 2, ventricular/cardiac muscle isoform |
| 17 | 4.91 | 19,116 | 28 | Myosin regulatory light chain 2, skeletal muscle isoform |
| 18 | 5.66 | 18,486 | 21 | Thiol-specific antioxidant enzyme |
| 20 | 5.33 | 12,835 | 51 | Chain A, a covalent dimer of transthyretin that affects the amyloid pathway |
| 21 | 5.34 | 15,048 | 34 | Galectin-1 |
| 22 | 6.03 | 11,073 | 66 | Haemoglobin beta chain |
| 23 | 6.03 | 11,073 | 37 | Haemoglobin beta chain |
| 24 | 4.97 | 21,665 | 31 | Myosin light chain 4 |
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