| Myosin amounts to more than 50% of myofibrillar protein in fish skeletal muscles, and as well a ubiquitous eukaryotic motor that interacts with actin to generate the force for cellular movements as diverse as cytokinesis and muscle contraction. Myosin consists of six subunits, two heavy chains and four light chains. It is divided into two functional domains, an N-terminal region called subfragment-1 (S1) forms a globular head, and contains ATP, and actin binding sites. The rod region shows a typical feature of anα-helical coiled-coil structure. The C-terminal half of rod called light meromyosin (LMM) is responsible for the thick filament formation, and has relationship with the gel-forming ability of myosin. Silver carp (Hypophthalmichthys molitrix), bream fish (Parabramis pekinensis), crucian carp (Carassius auratus) and bighead carp (Grammonoides opisthodom) are the most common cyprinid fish in the freshwater fish. Because these fish species belong to low-value and high-yielding freshwater fish, they are considered to be available for processing. So far, the molecular biological studies on freshwater fish muscle proteins were rarely reported. Accordingly, in the present study, the light meromyosins from these four types of freshwater fish were studied at the molecular level.In previous study, we have isolated two types of myosin heavy chain isoform gene from fast skeletal muscles of silver carp acclimatized in winter and summer by 3'-RACE, and named low temperature-type (sc-w) and high temperature-type (sc-s), respectively. However, these two isoform genes only encompassed about 550bp nucleotide sequence of 3'-terminal. The complete cDNAs encoding the two types of myosin heavy chain isoform have been also amplified by Long-PCR using their 3'-terminal sequence information. In this study, the full-length cDNAs encoding C-terminal half of myosin heavy chain, light meromyosin and deduced amino acid sequences were determined by extending 5′-terminals of the two known isoform genes using the two isoform genes complete cDNAs as templates. On the other hand, partial cDNA sequences of 3′-terminal for light meromyosin heavy chains from bream fish, crucian carp and bighead carp were cloned and sequenced by RT-PCR and 3′-RACE, which encompassed parts of 3′-translated and entire 3′-untranslated regions. The structures of these light meromyosins were were analyzed carefully by the bioinformatics software, and the genomic divergence and functional evolution of light meromyosin were further analyzed by means of phylogenetic tree.The results showed that the primary structure of complete LMM produced a 91.8% identity between sc-w and sc-s. Whereas a high identity of 96.9% was found between sc-w and gc10 which was reported to be isolated from 10oC-acclimated grass carp (Ctenopharyngodon idella). In contrast, the amino acid sequence of LMM for sc-s was much more similar to gcI (98.5%) and gc30 (97.1%) which were isolated from 30oC-acclimated grass carp. On the other hand, the amino acid sequence of partial LMM heavy chains for bream fish, crucian carp ,bighead carp ,which were obtained from winter, revealed high identity(97.1%-99.2%) to those of sc-w, gc10 and c10 which was reported to be isolated from 10oC-acclimated common carp (Cyprinus carpio). Comparing with the complete LMM isoforms from other animals, isoform-specific differences for sc-w and gc10 were clearly observed in 21 amino acid residues. Furthermore, among these amino acid mutations, 9 mutations occurred at the conserved residue sites. On the other hand, these two silver carp LMMs all had a characteristic seven-residue repeat (a, b, c, d, e, f, g)_n, where positions a and d were normally occupied by hydrophobic residues, however, the ratio of hydrophobic residue to the total was higher for the sc-s (31.1%) than sc-w (30.9%), suggesting that the former may form more stable coiled-coil ofα-helices than the latter type. The positions b, c e, f and g were occupied by charged residues, which were responsible for the thick filament formation. The deduced amino acid sequences of partial light meromyosin heavy chains from bream fish, crucian carp and bighead carp all showed a profile of hydrophilicity, but bream fish and bighead carp had higher hydrophilicity values than crucian carp in five regions. The results of the present paper demonstrated that these differences in the primary structures of the several types of cyprinid fish light meromyosin heavy chain isoforms might result in the differences in gel-forming profiles of myosins from fish in different seasons. According to the phylogenetic analysis, it seems that the close genetic distances were showed between the high temperature acclimated-freshwater fish, whereas the close genetic distances were showed between the low temperature acclimated-freshwater fish, which further demonstrated that the effects of environmental temperature on genomic divergence and functional evolution of fish myosin isoforms. |
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