Functional Basis of Three New Recessive Mutations of Slow Skeletal

Jul 18, 2016 - More TNNT1 NM mutations have been reported recently with similar recessive phenotypes. A nonsense mutation in exon 9 causes truncation ...
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Functional Basis of Three New Recessive Mutations of Slow Skeletal Muscle Troponin T Found in Non-Amish TNNT1 Nemaline Myopathies Chinthaka Amarasinghe, M. Moazzem Hossain, and J.-P. Jin* Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, United States ABSTRACT: Troponin T (TnT) is the tropomyosin (Tm)binding and thin filament-anchoring subunit of troponin and plays a central role in striated muscle contraction. A nonsense mutation in exon 11 of the TNNT1 gene encoding slow skeletal muscle troponin T (ssTnT) truncating the polypeptide chain at Glu180 causes a lethal recessive nemaline myopathy (NM) in the Amish (ANM). More TNNT1 NM mutations have been reported recently with similar recessive phenotypes. A nonsense mutation in exon 9 causes truncation at Ser108, and a splicing site mutation causes truncation at Leu203. Another splicing site mutation causes an internal deletion of the 39 exon 8-encoded amino acids. We engineered and characterized these ssTnT mutants to demonstrate that the Ser108 truncation exhibits a Tm binding affinity lower than that of the ANM Glu180 truncation, indicating a partial loss of Tm-binding site 1. Despite the presence of Tm-binding sites 1 and 2, ssTnT truncated at Leu203 binds Tm with decreased affinity, consistent with its recessive NM phenotype and the requirement of troponin complex formation for high-affinity binding of TnT to Tm. The exon 8-deleted ssTnT has a partial loss of Tm-binding site 1 but retains high-affinity Tm-binding site 2. However, exon 8-deleted ssTnT exhibits a dramatically diminished Tm binding affinity, indicating a long-range conformational effect of this middle region deletion. Predicted from the TnT structure−function relationship, removal of the N-terminal variable region partially rescued this negative impact. These novel findings lay a foundation for understanding the pathogenesis of TNNT1 myopathies and provide insights into the development of targeted treatment.

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Amish communities in Pennsylvania and Ohio. No effective treatment is currently available. The identification and mechanistic studies of ANM10−12 have raised clinical awareness and have prompted the inclusion of testing for TNNT1 mutations in the diagnosis of myopathies. As a result, more TNNT1 mutations have recently been reported in multiple non-Amish populations, which cause NM with recessive phenotypes similar to that of ANM.13−15 A nonsense mutation at codon Ser108 in the TNNT1 gene was found in a Hispanic patient in New York City.14 A Dutch nemaline myopathy patient was found to have compound heterozygote mutant alleles of TNNT1: a c.309+1G>A mutation at the invariant splice donor site of exon 8 causing aberrant RNA splicing to exclude the exon 8-encoded segment and a genomic deletion of the exon 14-encoded segment.13 A rearrangement in the TNNT1 gene (c.574_577 delins TAGTGCTGT) was found in nine Palestinian patients from seven unrelated families with recessively inherited NM.15 This mutation leads to aberrant splicing that causes a truncation of slow TnT at Leu203.

he TNNT1 gene encodes the slow skeletal muscle isoform of troponin T (TnT), one of the three subunits of the troponin complex.1 The troponin complex plays a central role in the allosteric functions of the striated muscle sarcomere by enacting conformational changes during the Ca2+-regulated contraction and relaxation.2−8 TnT is the thin filamentanchoring subunit of the troponin complex and is responsible for the incorporation of troponin into the sarcomeric thin filament. Three homologous TnT genes have evolved in vertebrates to encode muscle-type-specific isoforms expressed in cardiac, fast skeletal, and slow skeletal muscle fibers.1,9 TNNT1 nemaline myopathies (NM) reported to date represent a group of autosomal-recessive inherited myopathies caused by mutations in the TNNT1 gene, which prevent TnT from being incorporated into the myofilament, leading to progressive muscle degeneration and weakness.10−12 The first TNNT1 NM identified is the Amish NM (ANM) caused by a nonsense mutation in exon 11 of the TNNT1 gene, resulting in truncation of the slow skeletal TnT polypeptide chain at codon Glu180.10 ANM infants exhibit tremors and muscle weakness, followed by the development of contractures and progressive chest deformation due to weakness of the respiratory muscles. Death from respiratory insufficiency usually occurs in the second year. ANM has an incidence of 1 in 500 births in the © 2016 American Chemical Society

Received: June 7, 2016 Revised: July 13, 2016 Published: July 18, 2016 4560

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Figure 1. TnT constructs used in this study. (A) Intact ssTnT, ssTnT fragments, and ssTnT NM mutants were engineered, expressed in Escherichia coli, and purified. The middle fragment of ssTnT corresponds to the region between residues 51 and 151. The T2 fragment of ssTnT was prepared to represent the C-terminal region.4,38 Locations of the epitopes recognized by mAbs CT3,36 4B6, and 1G9 are indicated on the TnT construct maps. (B) SDS−PAGE gel and Western blots using mAbs CT3 and 4B6 for mapping the location of the epitopes recognized. (C) SDS−PAGE gel and Western blots of soleus, EDL, and heart muscles showed that while mAbs CT3 and T12 recognize slow/cardiac TnT and fast TnT, respectively, mAb 4B6 is specific to ssTnT.

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The three muscle-type TnT isoforms are highly conserved in the C-terminal and middle regions, reflecting conserved core structures that interact with troponin subunits C (TnC) and I (TnI), and contain two tropomyosin (Tm)-binding sites.1,16−18 The three TnT isoforms differ mainly in the regulatory Nterminal region, reflecting fiber-type-specific differentiations.1,19−21 X-ray crystallography resolved the atomic structure of a portion of the C-terminal region of TnT, which confirms the binding sites for TnC and TnI.8,22 However, no highresolution structure is available for TnT’s binding sites for Tm; thus, protein binding data and biochemical studies remain essential for understanding the interactions between TnT and Tm as well as the impact of pathogenic mutations. The TNNT1 NM mutations all have recessive phenotypes, providing novel and informative leads for understanding the structure−function relationship of TnT. In the study presented here, we engineered and characterized three new slow skeletal muscle TnT (ssTnT) mutations to compare with the ANM mutant. The results revealed unique impacts of the Ser108 and Leu203 truncations and the exon 8 internal deletion on the Tm binding affinity of TnT. The findings lay a foundation for understanding the pathogenesis of TNNT1 myopathies and provide new insights into the structure−function relationship of TnT to guide the development of targeted treatment.

EXPERIMENTAL PROCEDURES

Construction of Expression Plasmids and Preparation of Intact ssTnT and ssTnT Fragments. The representative ssTnT fragments and deletion mutants used in our study are outlined in Figure 1. cDNA-encoding fragments of human slow TnT were amplified by PCR from an intact human ssTnT cDNA and inserted into the pADE4 expression plasmid.23 After sequencing confirmation, the recombinant pAED4 expression vectors were used for the expression of intact and fragments of human ssTnT and the NM mutants in bacterial cultures. Competent BL21(DE3)pLysS Escherichia coli cells were transformed with the expression plasmid encoding intact ssTnT, ssTnT fragments, and mutants as previously described.12 Freshly transformed bacterial colonies were used to inoculate LB medium containing 100 μg/mL ampicillin and 12.5 μg/mL chloramphenicol. The cultures were incubated at 37 °C while being vigorously shaken and induced at an OD600 of ∼0.3 by adding IPTG to a final concentration of 0.4 mM. The cultures were continued for 3 h and harvested as previously described.12,24 Intact ssTnT and ssTnT1−204 fragment were purified via ionexchange chromatography and size-exclusion chromatography as previously described.20 ssTnT51−151 and ssTnT T2 fragments were purified via immobilized metal affinity chromatography as previously described. ssTnT-ΔE8, N-terminally truncated 4561

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KCl concentration (from 0.2 to 1.2 M). SDS−PAGE samples of the fractions were analyzed on a SDS−PAGE gel in Laemmli buffer as described previously.12,24 Solid-Phase Microplate Protein Binding Assay. An ELISA solid-phase microplate protein binding assay32 was used to compare the Tm binding affinities of the ssTnT constructs and mutants. Purified ssTnT or ssTnT fragments were dissolved in buffer A [100 mM KCl, 3 mM MgCl2, 1 mM EGTA, and 20 mM PIPES (pH 7.0)] at a concentration of 5 μg/mL. The ssTnT solution was added to 96-well polystyrene microtiter plates at a volume of 100 μL/well and incubated at 4 °C overnight to noncovalently immobilize the protein on the solid phase. The subsequent steps were performed at room temperature. Excess ssTnT proteins were removed by washing with buffer T (buffer A with 0.05% Tween 20) three times over a 10 min period. The plate was then blocked with buffer T with 1% bovine serum albumin (BSA) at room temperature for 1 h. Serial dilutions of rabbit skeletal muscle α/β-Tm in buffer T containing 0.1% BSA were added to the plate at a volume of 100 μL/well and incubated at room temperature for 2 h. The plates were washed four times with buffer T, and an anti-Tm mAb CH133 was added to the plate at a volume of 100 μL/well and incubated at room temperature for 1 h. After three buffer T washes, horseradish peroxidase-labeled goat anti-mouse secondary antibody (Santa Cruz) was added to the plate at a volume of 100 μL/well and incubated at room temperature for 45 min. After four buffer T washes, the amount of Tm bound to the immobilized ssTnT in each well was quantified via H2O2-ABTS [2,20-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] substrate colorimetric reaction. The A415 value in the linear course of the color development was monitored for each assay well with a reference wavelength of 655 nm using a Bio-Rad Benchmark automated microplate reader and recorded to plot Tm binding curves for each ssTnT construct. The experiments were conducted in triplicate wells and repeated. Reconstitution of the Troponin Complex and Tm Binding Assay. An ELISA solid-phase microplate protein binding assay was used to study the Tm binding affinities of the reconstituted troponin complex. Intact ssTnT was used to reconstitute the troponin complex as previously described.34 Rabbit skeletal muscle α/β-Tm was dissolved in buffer A at a concentration of 5 μg/mL. The Tm solution was added to 96well microtiter plates at a volume of 100 μL/well and incubated at 4 °C overnight to noncovalently immobilize the protein on the solid phase. The subsequent steps were performed at room temperature. Excess Tm was removed by washing with buffer T three times over a 10 min period. The plate was then blocked with buffer T with 1% BSA at room temperature for 1 h. Serial dilutions of the reconstituted troponin complex or ssTnT1−204 in buffer T containing 0.1 BSA + 1 mM DTT at pCa 4 or pCa 9 were added to the plate at a volume of 100 μL/well and incubated at room temperature for 2 h. After the plate had been washed four times with buffer T, anti-TnI mAb TnI-129 was added to the plate at a volume of 100 μL/well and incubated at room temperature for 1 h. After three washes with buffer T, a horseradish peroxidase-labeled goat anti-mouse second antibody (Santa Cruz) was added to the plate at a volume of 100 μL/well and incubated at room temperature for 45 min. After four more buffer T washes at pCa 4 or 9, the amount of troponin complex bound to the immobilized Tm in each well was quantified via H2O2-ABTS [2,20-azinobis(3-ethylbenzthia-

(ND) ssTnT-ΔE8, and ssTnT1−107 fragments were also purified via immobilized metal affinity chromatography with no additional residues at the N-terminus.25 Development of Slow TnT-Specific Monoclonal Antibodies. A short-term immunization procedure5 was applied to develop ssTnT-specific monoclonal antibodies (mAbs). Eightweek-old female Balb/c mice were immunized with 50 μg of purified mouse ssTnT1−204 fragment by intramuscular injection in 100 μL of phosphate buffered saline (PBS) mixed with an equal volume of Freund’s complete adjuvant. Ten days after the primary immunization, the mice were intraperitoneally boosted daily with 100 μg of the antigen in 200 μL of PBS without adjuvant on two consecutive days. Two days following the final boost, spleen cells were harvested from the immunized mouse to fuse with SP2/mIL-6 mouse myeloma cells with 50% polyethylene glycol 1500 (Invitrogen) containing 7.5% dimethyl sulfoxide as described previously.26,27 Hybridoma cells growing in 96-well culture plates were selected using HAT (0.1 mM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) medium containing 20% fetal bovine serum and screened by an indirect enzyme-linked immunosorbent assay (ELISA) using mouse ssTnT1−204 as the immobilized antigen and horseradish peroxidase (HRP)labeled, goat anti-mouse total immunoglobulin second antibody (Sigma). The positive hybridomas were subcloned three times using the method of limiting dilution to establish stable cell lines secreting mAbs against ssTnT. The immunoglobulin isotypes were determined using the hybridoma culture supernatant and a rat anti-mouse immunoglobulin isotyping ELISA kit (BD Biosciences) according to the manufacturer’s instructions. The specificity of the mAbs was determined using Western blotting against cardiac and skeletal muscle protein extracts as described previously.24 The following anti-TnT and anti-TnI mAbs with previously mapped epitope locations were also used in this study: CT3, a mouse mAb recognizing cardiac TnT and slow TnT;36 1G9, a mouse mAb specific to an epitope in the C-terminal T2 region of TnT;9 T12, a mouse mAb raised against fast TnT with weak cross-reaction with cardiac TnT;28 and TnI-1, a mouse mAb against TnI.29 SDS−PAGE and Western Blotting. Protein samples were homogenized in SDS−PAGE sample buffer containing 2% SDS, 0.3% bromophenol blue, 10% glycerol, 150 mM DTT, and 50 mM Tris-HCl (pH 8.8) and analyzed on gels in Laemmli buffer as described previously.12,24 The resulting gels were stained with Coomassie Blue R250 to reveal the protein bands. Duplicate gels were transferred to a nitrocellulose membrane, and Western blotting was performed as previously described.25 mAbs recognizing specific TnT epitopes9 were used to verify the intact ssTnT and ssTnT fragments used in our study. Tropomyosin Affinity Column Chromatography. Purified rabbit skeletal muscle α/β-Tm was prepared as previously described30 and covalently conjugated to CNBr-activated Sepharose 4B (GE Healthcare) as described.31 A Tm affinity column with a 0.5 mL bed volume was prepared and equilibrated in a buffer containing 50 mM KCl, 3 mM MgCl2, 20 mM PIPES (pH 8.0), 1 mM dithiothreitol, and 0.1 mM PMSF. A mixture of ssTnT and fragments dissolved in the column buffer was loaded onto the Tm affinity column. The column was then washed with the column buffer and eluted with the column buffer containing a series of step increases in 4562

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concentrations of KCl because of its loss of Tm-binding site 2 (Figure 1A). ssTnT1−204 representing the Leu203 truncation had a Tm binding affinity close to that of intact ssTnT and eluted at a high KCl concentration, reflecting the fact that ssTnT1−204 retains both Tm-binding sites 1 and 2 (Figure 1A). ssTnT S108ter Mutant Has a Tm Binding Affinity Lower Than That of the ANM E180Stop Mutant. The microplate solid-phase protein binding assay showed that ssTnT1−107 has a binding affinity for Tm significantly lower than that of the ANM E180ter mutation (Figure 3), indicating

zoline-6-sulfonic acid)] substrate colorimetric reaction. The A415 value reading and construction of Tm binding curves were performed as described above. The experiments were conducted in triplicate wells and repeated. Statistical Analysis. The absorbance values of each Tm binding curve were compared via two-way analysis of variance using GraphPad Prism. The means were compared across binding curves, and a Tukey’s multiple-comparison test was conducted with a 0.05 significance level. The free protein concentration for 50% maximal binding to the immobilized protein was obtained by fitting the sigmoidal curves with the least-squares method.

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RESULTS Engineered ssTnT and ssTnT Fragments and Specific Anti-ssTnT mAbs. The intact and representative fragments of ssTnT (Figure 1A) engineered and purified for our study are shown in the SDS−PAGE gel and verified in the Western blots using site-specific anti-TnT mAbs (Figure 1B). Among the new anti-ssTnT mAbs developed, 4B6 (IgG1κ) is highly specific to ssTnT without cross reaction to fast skeletal muscle and cardiac TnT (Figure 1C). Direct Comparison of Tm Binding Affinities of ssTnT and Mutants Using Affinity Column Chromatography. A mixture of intact ssTnT and NM mutants was analyzed on a Tm affinity column. The SDS−PAGE gel in Figure 2 shows the

Figure 3. ssTnT S108ter mutant that exhibited a Tm binding affinity significantly lower than that of the ssTnT E180ter ANM mutant. (A) Microplate protein binding experiments demonstrated Tm binding affinities for these two ssTnT mutants significantly lower than that of intact ssTnT. The S108ter mutant exhibits a nonsaturable Tm binding curve and an affinity for Tm much lower than that of the ssTnT E180ter mutant (**P < 0.005). (B) To verify equal coating of the ssTnT proteins on a microtiter plate, mAb CT3 (Figure 1A) ELISA titration showed very similar curves against the three coated ssTnT proteins. Figure 2. Direct comparison of Tm binding affinities of intact ssTnT and ssTnT NM mutants using column chromatography. TnT fragments representing the NM mutations were compared with intact ssTnT by analysis on a Tm affinity column. The SDS−PAGE gel shows the column loading and elution profile of the TnT proteins studied. The result shows that ssTnT fragments representing the exon 8 deletion and S108ter mutations exhibited drastically decreased Tm binding affinity, as both fragments eluted in the flow-through. The ANM E180ter mutation exhibited intermediate Tm binding affinity based on its intact Tm-binding site 1. In contrast, intact ssTnT and ssTnT1−204 fragment eluted at a much higher KCl concentration, consistent with the fact that they both retain the two Tm-binding sites (Figure 1).

that truncation at Ser108 partially destroyed Tm-binding site 1, whereas both S108ter and E180ter mutations exhibited a Tm binding affinity lower than that of intact TnT, reflecting the loss of Tm-binding site 2 (Figure 1A). The intact ssTnT and the E180ter mutant ssTnT both showed saturable binding, while the S108ter mutant exhibited a significantly lower affinity and a nonsaturable binding curve for Tm. ssTnT Leu203 Truncation Exhibits a Tm Binding Affinity Lower Than That of Intact ssTnT. A binary microplate solid-phase protein binding assay demonstrated that the ssTnT Leu203 truncation mutant exhibits a Tm binding affinity lower than that of intact ssTnT (Figure 4), despite its retention of both Tm-binding sites 1 and 2 (Figure 1A). The fact that the sensitivity of the solid-phase ELISA protein binding assay in detecting the decreased Tm binding affinity of the ssTnT Leu203 truncation is higher than that of Tm-affinity chromatography (Figure 2) reflects the effectiveness of stringent washes. The result indicates that the C-terminal distal

binding and elution profile of the ssTnT fragments in comparison with that of intact ssTnT. The result demonstrates that the ssTnT constructs representing the ΔE8 and S108ter mutants exhibited diminished Tm binding affinity, and both were present in the flow-through. The ssTnT E180ter mutant exhibited a low Tm binding affinity and eluting early at low 4563

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Figure 4. ssTnT1−204 exhibited a Tm binding affinity lower than that of intact ssTnT. Microplate protein binding experiments demonstrated that the ssTnT1−204 fragment representing NM ssTnT Leu 203 truncation had a Tm binding affinity lower than that of intact TnT (**P < 0.005). (B) To verify equal coating of the ssTnT proteins on the microtiter plate, mAb CT3 (Figure 1A) ELISA titration showed very similar curves against the two coated ssTnT proteins.

Figure 6. Internal deletion of the exon 8-encoded segment of ssTnT results in significantly weakened Tm binding. (A) Microplate protein binding experiments demonstrated that in contrast to that of intact ssTnT, the ssTnT ΔE8 mutant exhibited very low Tm binding affinity and a nonsaturable binding curve (**P < 0.005). (B) To verify equal coating of the ssTnT proteins on a microtiter plate, mAb 4B6 (Figure 1A) ELISA titration showed very similar curves against the two coated ssTnT proteins.

region of TnT may affect the Tm binding affinity via a conformational effect. The Tm Binding Affinity of the Reconstituted Troponin Complex Is Modulated by Calcium. The ssTnT Leu203 truncation mutant lacks the binding sites for TnI and TnC and, thus, is unable to form a tertiary troponin complex, which may contribute to decreased Tm binding affinity and the recessive phenotype that causes NM.15 Consistent with that seen in the fast skeletal muscle troponin complex,17,18 an increase in Tm binding affinity of the reconstituted slow skeletal muscle troponin complex was seen at pCa 4 in comparison to that at pCa 9 (Figure 5), supporting this hypothesis. The ssTnT ΔE8 Mutant Exhibits a Drastically Decreased Tm Binding Affinity. The microplate solidphase protein binding assay showed that the exon 8-deleted ssTnT had a very low binding affinity (Figure 6), despite the

presence of unaffected Tm-binding site 2 and only partial deletion of Tm-binding site 1 (Figure 1A). The results suggest that deletion of the exon 8-encoded segment may have a longrange conformational effect on the affinity of Tm-binding site 2. Removal of the N-Terminal Variable Region Partially Restores Exon 8-Deleted ssTnT’s Binding Affinity for Tm. It is known that deletion of the N-terminal variable region increases Tm binding affinity.35 Because this was hypothesized on the basis of the mechanism in which the N-terminal variable region of TnT modulates the TnT molecular conformation and decreases the Tm binding affinity,1 we tested the effect of its removal on rescuing the diminished Tm binding affinity of ΔE8 ssTnT. The microplate solid-phase protein binding assay showed that the removal of the N-terminal variable region significantly increased the Tm binding affinity of ΔE8 ssTnT (Figure 7). Although this restoration effect did not completely rescue the Tm binding affinity to the level of intact ssTnT or the T2 fragment of ssTnT (Figure 7), the results suggest a potential approach to rescuing the phenotype of the ΔE8 ssTnT mutation. The results further suggest that deletion of the exon 8encoded segment may augment the N-terminal modulation of the molecular conformation and function of the T2 region of ssTnT (Figure 8). Therefore, the data support that removal of the N-terminal variable region presents a potential therapeutic target for the treatment of ssTnT ΔE8 NM.

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Figure 5. Tm binding affinity of the reconstituted slow troponin complex at pCa 4 and 9. Microplate protein binding experiments demonstrated that calcium modulates Tm binding of slow skeletal muscle troponin with an affinity at pCa 4 higher than that at pCa 9 (**P < 0.005).

DISCUSSION TNNT1 encodes one of the two skeletal muscle TnT isoforms in vertebrates. Two alternatively spliced variants of ssTnT are normally expressed in human skeletal muscle.1,12 These splicing 4564

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Figure 8. Summary of the TNNT1 NM mutations. Illustrated on a structure−function model of the troponin complex (adapted from ref 22), the positions of ssTnT Ser108, Glu180, and Leu203 truncations and exon 8 deletion are indicated together with the interaction sites for Tm, TnI, and TnC. The long arrows propose the effects of the Nterminal variable region and the exon 8-encoded segment on the overall conformation and function of TnT. In this model, the effect of the N-terminal segment on decreasing Tm binding affinity was augmented by the internal deletion of the exon 8-encoded segment.

However, the results presented here show that the Tm binding affinity of the ssTnT S108ter mutant is significantly lower than that of the ANM ssTnT E180ter mutant (Figure 3) while both mutations delete Tm-binding site 2 (Figure 1A). Considering that the ssTnT E180ter mutant binds Tm with binding site 1 intact (Figure 1A), the new observation indicates that the truncation at Ser108 damages Tm-binding site 1. The data demonstrate that the middle region of Tm-binding site 1 extends beyond the segment of amino acids 64−108, although the exact downstream boundary remains to be determined. We previously demonstrated that ANM muscle biopsies had no detectable ssTnT E180ter mutant protein; thus, it is not incorporated into the myofilament.11 Therefore, the more severely diminished Tm binding affinity of the ssTnT S108ter mutant should also result in no myofilament incorporation, consistent with the recessive nemaline myopathy phenotype of the patient.14 Negative Impact of the C-Terminal Truncation on the Tm Binding Affinity of the ssTnT Leu203 Truncation Mutant. Although the Leu203 truncation mutant contains both Tm-binding sites 1 and 2 (Figure 1A), it had a Tm binding affinity lower than that of intact ssTnT that could be detected using the binary solid-phase protein binding assay (Figure 4). This result demonstrates that the removal of the C-terminal portion of ssTnT after Leu203 has a negative impact on the binding affinity for Tm. One hypothesis is that the deletion of the C-terminal segment may have effects on the overall conformation of TnT, as TnT is an allosteric protein and known to undergo conformational modulations that have been extensively shown with N-terminal modifications.5,7,8,36 Tm binding of rabbit fast skeletal muscle TnT has been shown to be affected by calcium in studies using the tertiary troponin complex.16,17 Our study also showed that Tm binding of the troponin complex reconstituted using intact ssTnT is also dependent on calcium; a higher affinity was found at pCa 4 than at pCa 9 (Figure 5). The ssTnT Leu203 truncation mutant lost the binding sites for TnI and TnC (Figure 1A) and, thus, is unable to form the troponin complex. The lack of this calciumenhanced Tm binding might be one of the factors contributing to a loss of myofilament incorporation and the recessive NM phenotype of the Leu203 truncation mutation.15 It would be interesting to confirm this hypothesis by examining the TnT

Figure 7. Removal of the N-terminal variable region partially rescues the Tm binding affinity of exon 8-deleted ssTnT. Microplate protein binding experiments demonstrated that the ssTnT ΔE8 mutant exhibited very low Tm binding affinity and a nonsaturable binding curve, even significantly weaker than that of the T2 fragment of ssTnT (**P < 0.005). Removal of the N-terminal segment of the exon 8deleted ssTnT led to a partial but highly significant restoration of the Tm binding affinity (**P < 0.005). (B and C) To verify equal coating of the ssTnT proteins on microtiter plate, mAb 4B6 and 1G9 (Figure 1A) ELISA titrations, respectively, showed very similar curves against the coated ssTnT proteins.

variants differ by inclusion or exclusion of the exon 5-encoded segment in the N-terminal region.12 The TNNT1 NM mutations reported to date are all located downstream of this alternatively spliced segment. Therefore, the mutants could be in both splice forms. Indeed, we have found that the ANM mutant mRNA contains both splice forms.12 It is intriguing that the loss of only ssTnT causes very severe NM. Understanding the pathogenesis of TNNT1 myopathies is essential for the development of targeted treatment. Via characterization of the function of the three newly identified TNNT1 NM mutants, the results of our study present several novel findings that provide new insights into the pathogenic mechanisms and the structure−function relationship of TnT. S108ter Truncation of ssTnT Damages Tm-Binding Site 1. In previous work, the first Tm-binding site has been localized to the middle region of TnT and was hypothesized to be within the region of amino acids 64−102 in slow TnT.24,25 4565

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around the globe, e.g., the S108ter, exon 8 deletion, and Leu203 truncation mutations investigated in this study, demonstrates that TNNT1 myopathy is no longer an isolated disease in the Amish. This trend demonstrates that as exome sequencing becomes increasingly common in the clinical diagnosis of genetic disorders, identification of more myopathic mutations in the skeletal muscle TnT genes can be anticipated. While TNNT1 mutations are recognized as one of the prime genetic causes of skeletal muscle myopathies, characterization of the Tm binding function of the three new TNNT1 NM mutations establishes the molecular basis for the various structural disruptions in different regions of the ssTnT polypeptide chain to lead to loss-of-function recessive phenotypes. Although we investigated only the effects of the exon 8 deletion, the clinical case report found it in a compound heterozygote of ssTnT exon 8 deletion and exon 14 truncation.13 Therefore, we anticipate that truncation of the exon 14-encoded C-terminal end segment also results in a lossof-function phenotype, rendering the recessive NM phenotype of the compound heterozygote patient. While this hypothesis remains to be investigated, our preliminary data (not shown) indicate that deletion of the C-terminal end segment of TnT may result in instability of the TnT protein and significantly decrease the Tm binding affinity to hamper incorporation into the thin filament. By systemic characterization of four representative TNNT1 NM mutants to elucidate the key structures in the TnT polypeptide underlying their conformational and functional impacts, this study paves the way to a better understanding of the pathogenesis of TNNT1 NM and provides novel information for the development of targeted treatments of these lethal diseases.

protein contents in the muscle of patients with the c.574_577 delins TAGTGCTGT mutation or animal models expressing the C-terminally truncated ssTnT1−203. Direct and Indirect Negative Impacts of Deleting the Exon 8-Encoded Segment of ssTnT. On the basis of the known structure−function relationship of TnT, exon 8-deleted ssTnT is anticipated to have a partially destroyed Tm-binding site 1, whereas Tm-binding site 2 remains intact (Figure 1A). The fact that the ssTnT ΔE8 mutant exhibited a very low affinity and a nonsaturable Tm binding curve, which is significantly weaker than that of the ssTnT T2 fragment containing only Tm-binding site 2, was unexpected (Figures 6 and 7). The data suggest that the internal deletion of a segment in the middle region of ssTnT may have not only damaged Tmbinding site 1 but also had a long-range conformational impact on downstream Tm-binding site 2 (Figure 8). This negative effect appears to be very predominant, diminishing the Tm binding affinity of the ssTnT ΔE8 mutant to a level significantly lower than that of the S108ter and ANM S180ter mutants (Figures 3 and 7). N-Terminal Deletion-Based Conformational Modulation Rescues the Tm Binding Affinity of the ssTnT ΔE8 Mutant. The N-terminal variable region of TnT is a regulatory structure that does not bind to any other known thin filament proteins but causes overall conformational changes in TnT, fine-tuning myofilament functions and muscle contractility.1 Alternative RNA splicing and restrictive proteolysis are two mechanisms that modify the N-terminal structure of TnT and modulate molecular conformation and function.1 The most investigated example is the restrictive N-terminal truncation of cardiac TnT during myocardial adaptation that improves cardiac efficiency in ischemia and left ventricular pressure overload.37 At the level of protein interactions, the deletion of the Nterminal variable region of TnT increases the Tm binding affinity.31 In the study presented here, the deletion of the Nterminal variable region of ΔE8 ssTnT produced a partial but significant restoration of Tm binding affinity (Figure 7). This finding not only confirms the effect of the N-terminal variable region of TnT on reducing Tm binding affinity but also suggests two possible mechanisms in the context of TnT structure−function relationships. The first is that the Nterminal segment highly effectively modulates the conformation and function of TnT by influencing the middle and C-terminal T2 regions (Figure 8). The second is that the internal deletion of the exon 8-encoded segment in the middle region of ssTnT not only causes a partial destruction of Tm binding site 1 but also augments the effect of the N-terminal region on decreasing the affinity of Tm-binding site 2, likely by facilitating the transmission of the allosteric modulation. These novel findings demonstrate that N-terminal modification or deletion may be further investigated as a potential therapeutic target for restoring the Tm binding affinity of the ssTnT ΔE8 mutant and other myopathic TnT mutations. TnT Myopathies Revisited. Whereas numerous missense and single-residue deletion mutations in the TNNT2 gene encoding cardiac TnT have been reported to cause cardiomyopathies, very few myopathic mutations have been reported in the fast (TNNT3) and slow (TNNT1) skeletal muscle TnT genes.1 TNNT1 myopathy was first found in the Old Order Amish in Pennsylvania, leading to identification of the ANM E180ter nonsense mutation.10 The finding of additional TNNT1 NM mutations in multiple ethnic groups

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AUTHOR INFORMATION

Corresponding Author

*Department of Physiology, Wayne State University School of Medicine, 5374 Scott Hall, 540 E. Canfield, Detroit, MI 48201. Telephone: 313-577-1520. Fax: 313-577-5494. E-mail: jjin@ med.wayne.edu. Funding

Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health via Grant AR048816 to J.-P.J. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Ms. Hui Wang for technical assistance. ABBREVIATIONS ANM, Amish nemaline myopathy; BSA, bovine serum albumin; cDNA, complementary DNA; cTnT, cardiac troponin T; EGTA, ethylene glycol tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; fsTnT, fast skeletal troponin T; IPTG, isopropyl β-D-1-thiogalactopyranoside; mAb, monoclonal antibody; NM, nemaline myopathy; PCR, polymerase chain reaction; PMSF, phenylmethanesulfonyl fluoride; SDS− PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; ssTnT, slow skeletal troponin T; Tm, tropomyosin; Tn, troponin complex; TnC, troponin C; TnI, troponin I; TnT, troponin T. 4566

DOI: 10.1021/acs.biochem.6b00577 Biochemistry 2016, 55, 4560−4567

Article

Biochemistry

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DOI: 10.1021/acs.biochem.6b00577 Biochemistry 2016, 55, 4560−4567