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

Abstract

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.

Recent advances in cattle functional genomics and their application to beef quality

“…The levels of expression of the other six were unchanged or decreased in DM and H muscles. These fTnT isoforms differ in terms of the presence of the alternative splicing region corresponding to mutually exclusive exons 16 and 17 , Muroya et al, 2003. All these data demonstrated that the TnT fast isoforms over-expressed in the muscle of high muscle growth potential (DM and H) contained the exon 16 and the others contain the exon 17.…”

Proteome dynamics during contractile and metabolic differentiation of bovine foetal muscle

“…These two isoforms are grouped in cluster 5, which displayed up-regulated proteins at 260 dpc. Fast TnT isoforms proteins are also the product of a complex alternative splicing (Muroya et al, 2003;Bouley et al, 2004). Our results displayed four different isoforms of fTnT according to their pI and MW.…”

“…According to these authors, these two slow TnT isoforms are the product of alternative splicing of the murine exon 5 (Huang et al, 1999). The most acidic one is referred to as sTnT1 by Muroya et al (2003) and the most basic one is referred to as sTnT2. These two isoforms are grouped in cluster 5, which displayed up-regulated proteins at 260 dpc.…”

Identification of small troponin T peptides generated in dry-cured ham

“…In this way, Whipple and Koohmaraie (1992) examined the amount of post-mortem proteolysis in three different lamb muscles and concluded that degradation products derived from TnT in supraspinatus, a slow type muscle, were distinct from those in longissimus and gluteus medius, fast type muscles. On the other hand, Muroya, Nakajima, and Chikuni (2003), established that slow-oxidative muscles were richer in slow TnT isoforms than were the fast muscles. In this study, 25 fragments of fast TnT have been identified against only two slow TnT fragments.…”