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Amino acid sequences of multiple fast and slow troponin T isoforms expressed in adult bovine skeletal muscles

Department of Animal Products, National Institute of Livestock and Grassland Science,Tsukuba, Ibaraki 305-0901, Japan

¹ Correspondence:
(phone: +81-0-29-838-8686; fax: +81-0-29-838-8683; E-mail:
muros{at}affrc.go.jp).

	Abstract

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Multiple nucleotide sequences of complementary DNA (cDNA) of bovine troponin T (TnT) isoforms expressed in the adult skeletal muscles were determined to facilitate the elucidation of the TnT degradation progress during postmortem aging of muscles. Fresh muscle samples were excised from the lingual, masseter, pectoralis, diaphragm, psoas major, longissimus thoracis, spinnalis, semitendinosus, semimembranosus, and biceps femoris muscles of three Holstein cows within 1 h of slaughter. Complementary DNA fragments of fast and slow TnT isoforms expressed in each muscle were amplified by reverse-transcribed PCR. Consequently, four major fragments of fast TnT and two fragments of slow TnT, all of which contained the complete coding region, were obtained. The sequence determination of these fragments revealed that at least eight and two isoforms were generated by the alternative splicing from bovine fast and slow TnT messenger RNA, respectively. In the fast TnT isoforms, five small variable exons were observed; three of these five exons were in the amino (N)-terminal region. The calculated molecular weight of fast and slow TnT isoforms ranged from 29,816 to 32,125 and from 30,166 to 31,284, respectively. The deduced amino acid sequences revealed that the N-terminal region of all the TnT isoforms was extremely glutamic acid-rich. Reverse-transcribed PCR analysis revealed that expression of each of these isoforms was distributed in a fast or slow muscle-specific manner. Given that TnT degradation has been reported to accompany a decrease in glutamic acid content in the conventional 30-kDa degradation product, the sequence data suggested that the 30-kDa fragment seem to be generated by the proteolytic removal of the glutamic acid-rich N-terminal ends. The multiplicity of TnT isoforms may result in a complicated pattern of TnT degradation on SDS-PAGE gel during beef aging.

Key Words: Aging • Amino Acid Sequence • Cattle • Degradation • Troponins

	Introduction

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Troponin T (TnT) is an important regulatory and structural component of skeletal muscle thin filaments (Perry, 1998). In bovine muscles, at least two TnT isoforms are present (Clarke et al., 1976). From the fast and slow TnT genes, multiple TnT isoforms are generated by alternative splicing of the messenger RNA in avian and mammalian species (Perry, 1998).

In postmortem bovine muscle, a 30- or 32-kDa peptide, which was immunologically identified as a TnT fragment (Ho et al., 1994; Huff-Lonergan et al., 1996; Negishi et al., 1996), is the first degradation product of a skeletal muscle component that is associated with meat tenderness (MacBride and Parrish, 1977; Olson and Parrish, 1977). The degradation of TnT progresses simultaneously with the postmortem tenderization of beef, indicating a strong correlation between the two events (Penny and Dransfield, 1979). However, the details of TnT degradation remain poorly understood. For a better understanding of the degradation, it is necessary to determine the amino acid sequence of bovine TnT isoforms on which the 30 kDa can be mapped. Moreover, since bovine muscles express various fast and slow TnT isoforms, as we reported recently (Muroya et al., 2002a), it is expected that there is considerable variation in the TnT degradation originated from the isoforms. The presence of the isoforms often complicates the SDS-PAGE band pattern (Ho et al., 1994; Huff-Lonergan et al., 1996). Therefore, for purposes of identifying the types of peptides on SDS-PAGE, it is also necessary to comprehensively determine the TnT isoform types that are expressed in adult bovine skeletal muscles.

This study was performed for the purpose of understanding the degradation of TnT and the differences among the isoforms. We determined complementary DNA (cDNA) sequences of TnT isoforms expressed in adult bovine skeletal muscles in this work. The distribution of TnT isoforms among bovine muscles is also described in this paper.

	Materials and Methods

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Treatment of Animals and Sample Preparation
Animals were cared for as outlined in the Guide for the Care and Use of Experimental Animals (Animal Care Committee of the National Institute of Livestock and Grassland Science, Japan). Three Holstein cows aged 66 to 95 mo were slaughtered after captive-bolt gun stunning. Ten skeletal muscles, namely the lingual (TN), masseter (MS), pectoralis (PP), diaphragm (DP), psoas major (PM), longissimus thoracis (LT), spinnalis (SP), semitendinosus (ST), semimembranosus (SM), and biceps femoris (BF) muscles, were excised from each cow within 1 h after slaughter. Immediately, approximately 1-cm³ pieces from the central part of each muscle were prepared and frozen in liquid nitrogen. The frozen samples were then crushed into fine powder in a liquid nitrogen cold crusher (NRK R-8; Nihon-Rikagakukikai, Tokyo, Japan).

Messenger RNA Preparation and Complementary DNA Synthesis
Muscle samples taken from each cow were dissolved in ISOGEN, a total RNA extraction kit (NipponGene, Tokyo, Japan), and total RNA was extracted from each muscle according to the manufacturer’s protocol. The first-strand cDNA was synthesized from 2 µg of total RNA in 40 µL of reaction mixture containing 400 U of M-MLV reverse transcriptase RNase-H minus (Toyobo, Tokyo, Japan), 1 mM deoxynucleoside triphosphate, 1 µM 3ADP1T, 5 ' - C T G C A G G A A T T C G A T A T C G A A G C T T G C - ( T ) ₁₅ ( A / C / G ) (A/C/G/T)3' (Chikuni et al., 2001). The reaction mixture was incubated at 42°C for 1 h.

Polymerase Chain Reaction Analysis and Nucleotide Sequence Determination of Complementary DNA
The sequences of primers used for troponin T sequence determination are shown in Table 1. At first, the TnT primers were designed basically according to partial bovine fast and slow TnT (accession numbers BG223670 and BF261276 in the DDBJ/EMBL/GenBank nucleotide sequence databases, respectively) and human fast and slow TnT (accession numbers NM_006757 and NM_003283, respectively) sequences.

Prediction of Molecular Properties from Complementary DNA Sequence
The AA sequences of the TnT isoform proteins were deduced from the nucleotide sequences of the cDNA by Genetyx-Mac (Ver. 9.0, Software Development Co., Ltd., Tokyo, Japan). The predicted molecular weight (MW) and isoelectric point (pI), using the method of Skoog and Wickman (1986), were also calculated by Genetyx-Mac.

Determination of Troponin T Isoform Types Expressed in Muscles
Expression of fast and slow TnT in 10 muscles of each of three cows was analyzed by reverse-transcribed (RT)-PCR using various primer combinations. The primer sequences (Table 1) were designed according to the bovine sequences determined in this study. The PCR procedures with AmpliTaq Gold (Perkin-Elmer) were carried out as previously described using synthesized cDNA of each muscle as the template. For determination of the fast TnT isoform types expressed in each muscle, first a common primer f2 and an exon 16- or 17-specific primer (16R2 and 17R1, respectively) were used (Figure 1a). Then, for each PCR product, common primers f2 and fR3 or 9R were used to detect isoforms of the amino (N)-terminal variable region. For the slow TnT type determination, common primers s1 and sR4 were used (Figure 1b). Multiplex PCR for comparison of expression of fast and slow TnT was also performed using f5 and f16R, and s5 and sR2 primers for amplification of fast and slow TnT fragments, respectively.

View larger version (9K): [in this window] [in a new window]   Figure 1. Panels give the location of primers used in this study on complementary DNA of bovine fast troponin T (fTnT; panel a) and slow troponin T (sTnT; panel b). Primers are indicated as filled boxes with arrowheads. The number in the open box indicates the putative number of each exon.

	Results

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Full-Length Amino Acid Sequences of Bovine Fast and Slow Troponin T Isoforms
The multiple sequences of the bovine fast TnT isoforms revealed that all the TnT isoforms have at least 13 exons in the coding regions (Figure 2a), which was predicted by comparison with the other fast TnT, and that three of five variable exons existed in the N-terminal region. The three N-terminal exons corresponded to exons 4, 7, and 8 of murine fast TnT (Breitbart and Nadal-Ginard, 1986; Jin et al., 1997). The number and boundaries of exons in the bovine fast TnT sequence were predicted based on the identity between the bovine cDNA and the rat fast TnT gene (Breitbart and Nadal-Ginard, 1986). According to the rat fast TnT gene sequence, it is more likely that bovine peptide APPPP corresponds to rat APEP, which is encoded in the exon 7 (Figure 3). The bovine-specific peptide AEVPEVHEEVHEVHEP was shown to be a part of bovine exon 7 by PCR amplification within the exon using genomic DNA as the template (data not shown).

View larger version (92K): [in this window] [in a new window]   Figure 2. Amino acid sequences of the longest bovine fast troponin T isoforms (fTnT1/16 and 17; panel a) and slow troponin T isoforms (sTnT1 and sTnT2; panel b). The AA sequences were deduced according to the result of complementary DNA sequence determination. Closed and open arrowheads indicate actual and putative exon boundaries, respectively. The number above the sequence indicates the putative number of each exon. Underlined peptide is characteristic to bovine fTnT.

View larger version (27K): [in this window] [in a new window]   Figure 3. Amino acid sequence identity of fast troponin T N-terminus among mammals and avians. The N-terminal amino acid sequence was compared among bovine, human (Wu et al., 1994), rabbit (Fujita et al., 1990; Briggs and Schachat, 1993), rat (Medford et al., 1984; Breitbart et al., 1985; Breitbart and Nadal-Ginard, 1986), mouse (Wang and Jin, 1997), chicken (Smillie et al., 1988), and quail (Bucher et al., 1999). Amino acid residues shaded with black or gray are conserved among all or some species shown in the alignment, respectively.

In the AA sequences, the N-terminal region of bovine fast TnT was a highly glutamic acid-rich sequence (Figure 2a), which is also the case in other mammalian and avian fast TnT. More than 30% of the total glutamic acid residues of each fast TnT sequence were present in the region between the N-terminus and exon 8, where no lysine or arginine residues were present. Likewise, the N-terminal region of the slow TnT was also rich in glutamic acid residues but had no lysine or arginine residues (Figure 2b).

By comparison of the fast TnT AA sequence that contains all exons, the overall identity of the adult type bovine proteins with those of other species was more than 74% (Table 2). However, the N-terminal region is highly variable among vertebrates (Figure 3), whereas the identity between the other bovine region and those of other species was more than 85%. The most characteristic region of the bovine fast TnT primary structure was AA 28-43, which was similar to the rabbit sequence 27-39, but which could not be found in murine or human proteins (Figure 3). These AA sequence data revealed that the N-terminal region of fast TnT reflects the species specificity of the fast TnT molecule.

Exon Combination and Predicted Molecular Properties of Multiple Troponin T Isoforms
By subsequent sequence determination, it was confirmed that eight fast TnT (fTnT1/16 or 17, fTnT2/16 or 17, fTnT3/16 or 17, and fTnT4/16 or 17) and two slow TnT (sTnT1 and sTnT2) isoforms were expressed in bovine muscles (Table 3). The nucleotide sequence data reported in this paper appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the sequential accession numbers AB085592 through AB085601.

The deduced AA sequences of the fast TnT isoforms showed different predicted MW and pI among the proteins (Table 3). The MW of the fast and slow TnT isoforms ranged from 29,816 to 32,125 and from 30,165 to 31,283, respectively. Based on the pI, the fast TnT isoforms diverged into two distinct classes: acidic isoforms with pI ranging from 5.87 to 6.51 (fTnT1/16 or 17 and fTnT/16 or 17) and basic isoforms with pI ranging from 8.69 to 9.23 (fTnT3/16 or 17 and fTnT4/16 or 17). On the other hand, both of the two slow TnT isoforms were acidic proteins (pI 5.61 to 6.20; Table 3). The low pI of the two isoforms is due to their richness in glutamic acid residues in the N-terminal region including the exon 5.

Distribution of Fast and Slow Troponin T Isoforms Among Bovine Muscles
The expression of each TnT isoform in the 10 bovine muscles was detected as a band corresponding to that of MS or LT and DP or BF on the RT-PCR result for fast and slow TnT, respectively. According to the results, fTnT1 isoforms were expressed in TN, MS, and DP, but not in PP, PM, LT, SP, ST, SM, and BF, whereas fTnT3 isoforms were expressed in the muscles that lacked fTnT1 isoforms expression (Table 4). The fTnT2 isoforms were expressed in the all muscles except TN and MS. The fTnT4 isoforms were expressed in TN, MS, DP, and SP, but not in the other muscles. Thus, fTnT1 or fTnT4 isoforms and fTnT2 or fTnT3 isoforms demonstrated mutually exclusive expression among the bovine muscles. On the other hand, sTnT1 was expressed in the all muscles tested, whereas sTnT2 was expressed only in TN, DP, and SP (Table 4). The sTnT2 expression pattern was similar to that of fTnT1 and fTnT4 isoforms.

View larger version (31K): [in this window] [in a new window]   Figure 4. Ratio of expression of fast troponin T (fTnT) to slow troponin T (sTnT) isoforms. Multiplex reverse transcriptase-PCR of fTnT and sTnT isoforms was performed and results revealed the same expression pattern (TN = lingual muscles; MS = masseter; PP = pectoralis; DP = diaphragm; PM = psoas major; LT = longissimus thoracis; SP = spinnalis; ST = semitendinosus; SM = semimembranosus; BF = biceps femoris).

	Discussion

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Although the N-terminal AA residue of the degradation product remains undetermined, Negishi et al. (1996) reported that a decrease in the acidic properties of troponin complex accompanies TnT degradation. Further, the AA composition analysis of both intact and degraded TnT peptide showed that a decrease in the content of glutamic acid residue and increases in the content of both lysine and arginine residue in TnT occurred, accompanying the degradation of TnT into the 32 kDa that was thought to correspond to the conventional 30 kDa. On the other hand, the deduced AA sequences of multiple bovine TnT isoforms determined in the present work revealed that several clusters of glutamic acid residue were present in the N-terminal region of all the isoforms, whereas almost all lysine and arginine residues were located in the rest of the C-terminal side of each isoform. If the 30-kDa fragment is generated by a reduction of the N-terminal region, such changes in the AA composition are expected to occur. In our preliminary Western blot analysis using LT myofibril, degradation of intact TnT into the major fragment which corresponds to the conventional 30 kDa accompanied a reduction of 4 kDa (data not shown), which does not contradict with the reduced molecular weight observed in previous studies (Ho et al., 1994; Huff-Lonergan et al., 1996; Negishi et al., 1996). Among TnT isoforms, fTnT2/16 and 17, and fTnT3/16 and 17 were almost exclusively expressed in bovine LT. In such a case of isoform degradation, the first 35 AA residues corresponding to the reduced weight of approximately 4 kDa are expected to be removed from the intact TnT isoforms, which would result in a 3.6 to 4.6% decrease in the content of glutamic acid residue. This presumptive reduction of glutamic acid residues from TnT was in good agreement with the 4.2% decrease in its content found during generation of the 32 kDa (Negishi et al., 1996).

Although the detailed steric structure of TnT protein has not been determined so far, the strongly acidic N-terminus of TnT is probably extended and must be available to solvent because the Ser₁ is readily phosphorylated by TnT- and phosphorylase b-kinases (Perry, 1998). This suggests that, due to the large clusters of glutamic acid residues, the hydrophilic N-terminus can be attacked easily by proteases if its structure remains intact. Taken together, these data indicate that the degradation of bovine TnT during postmortem beef aging probably progresses by a reduction of amino acids of the N-terminal region.

On the other hand, by using an in vitro system, Hughes et al. (2001) demonstrated that rabbit TnT is initially cleaved at both the two N-terminal sites and the two C-terminal sites by bovine µ-calpain, which was determined by N-terminal sequencing and mass spectrometry. However, their SDS-PAGE analysis revealed that the molecular mass of each peptide generated by the proteolysis was no more than 26 kDa. Moreover, because the substrate of µ-calpain was a purified TnT, but not myofibril-including TnT, the attack of µ-calpain on TnT might be different from that in vivo (Hughes et al., 2001). Although µ-calpain is a good candidate that degrades TnT, the cleavage pattern analyzed by Hughes et al. (2001) is not likely to reflect TnT degradation in vivo.

The AA sequence of N-terminal exons of fast TnT differs among bovine, human, rabbit, rat, mouse, chicken, and quail, suggesting that TnT has species-specific physiological characteristics. Nevertheless, in the primary structure of both fast and slow TnT, in particular, the glutamic acid-rich N-terminal region and the constitutive exons were well conserved. This suggests that the function for muscle contraction is, to some extent, evolutionally conserved in both isoforms. Possibly due to its well-conserved structure, TnT can be degraded in the typical manner, which results in generation of the 30-kDa peptide during postmortem aging of the muscles. Actually, postmortem proteolysis in bovine, lamb, and porcine longissimus generated the 30 kDa peptide in a highly similar manner (Koohmaraie et al., 1991). In addition, an in vitro degradation study revealed that the respective degradation patterns of rabbit, chicken, and bovine TnT by lysosomal enzymes were quite similar (Mikami et al., 1987).

Possible Implication of Multiplicity of Bovine Troponin T Isoforms in the Degradation Pattern
The presence of multiple bovine TnT isoforms has long been known (Clarke et al., 1976). The isoforms are often found as intact proteins and as fragments on SDS-PAGE gels during postmortem aging of bovine muscles (Ho et al., 1994; Huff-Lonergan et al., 1996; Hughes et al., 2001). The multiplicity of the isoforms can complicate the TnT degradation pattern on the SDS-PAGE gel because determining whether the fragments detected by anti TnT antibodies are intact or degraded is sometimes difficult to distinguish. For example, Negishi et al. (1996) concluded that a 34-kDa component, appearing below the intact TnT band, was a degradation product; however, that component decreased during postmortem aging as the intact TnT was degraded in a similar manner and was accompanied with an increase of the 32-kDa degradation product. Therefore, the 34-kDa component might have been an intact TnT isoform. Furthermore, appearance of a degradation product might have been overlapped on the 34-kDa component. Actually, a band right below the intact TnT was also immunologically detected by Huff-Lonergan et al. (1996), but was concluded to be an isoform because its behavior was highly similar behavior to that of the intact TnT band.

In the present study, multiple TnT isoform messenger RNA were detected, and differences were found among them in terms of the deduced AA sequence, MW, and pI. Some of them had highly similar MW, which can make it difficult to distinguish the bands from each other and/or from that of the degradation product. Moreover, the degradation may vary among the isoforms as a result of differences in the AA sequence and pI. Difference in phosphorylation may also contribute to the appearance of the isoforms on SDS-PAGE gels. For a more thorough understanding of the appearance of TnT fragments, the TnT bands on SDS-PAGE gel should be carefully identified with consideration given to the TnT isoforms. Both the intact and the degraded peptides of bovine TnT can be precisely identified by further N-terminal determination of the peptides combined with the present AA sequences.

Muscle-Specific Expression of Each Troponin T Isoform in Bovine Muscles
Reverse-transcribed PCR analysis revealed that each of bovine TnT isoforms was distributed in a muscle-specific manner, although the ratio of expression in each muscle among fast or slow TnT isoforms could not be determined. In faster muscles, both fTnT2 and fTnT3 isoforms were predominantly expressed, but expression of sTnT1 isoform was also detected. On the other hand, in slower muscles, sTnT1 and sTnT2 isoforms were predominantly expressed, but fTnT1 and fTnT4 isoforms were also detected. In spite of differences in AA sequences of TnT isoforms among animals, muscle-specific distribution patterns of the isoforms have also been reported in rat (Breitbart et al., 1985) and rabbit muscles (Briggs et al., 1987). The expression pattern of TnT isoforms is consistent with that of fast and slow myosin heavy-chain isoforms in bovine muscles (Muroya et al., 2002b), suggesting that the expression and alternative splicing of TnT are regulated in a fast or slow muscle-specific manner. Since these TnT isoforms tend to be restricted to functionally different fast-glycolytic, fast-oxidative glycolytic, or slow-oxidative muscle fibers (Moore et al., 1987), the muscle-specific distribution pattern may indicate the ratio of each fiber among the muscles and the distinct physiological property of each muscle. The postmortem degradation products of TnT may also differ among the muscles both qualitatively and quantitatively; therefore, it is necessary to identify the TnT degradation products in both fast and slow muscles based on the AA sequence study.

	Implications

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Multiple nucleotide sequences of complementary DNA of bovine troponin T isoforms were determined. The sequence determination revealed that the various combinations of small exons in the amino acid-terminal region generated a multiplicity of isoforms (eight fast and two slow isoforms) that were expressed in adult bovine muscles. The deduced amino acid sequences revealed that the N-terminal region of all the troponin T isoforms was extremely rich in glutamic acid. Given that the troponin T degradation was reported to accompany a decrease in glutamic acid content in the major degradation product, the sequence data suggested that degradation of the product starts at the N-terminal region. The multiplicity of the troponin T isoforms may result in a more complicated pattern of degradation during postmortem beef aging than previously thought.

Received for publication September 23, 2002. Accepted for publication January 22, 2003.

	Literature Cited

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