Expression and functional analyses of two essential myosin light chains from the fast white muscle of the spotted mandarin fish Siniperca scherzeri

Abstract

Objective: The essential myosin light chains (EMLC) are the major structural and regulatory proteins that control actin-myosin interaction in striated muscles. The purpose of this work is to provide a better understanding about the EMLC properties and their expression pattern during muscle development in the mandarin fish. Material and Methods: The cDNA library was constructed to isolate and characterize MLC sequences. The nucleotide sequences and inferred amino acid sequences were analyzed. Whole-mount in situ hybridization was carried out to investigate the gene expression pattern. To assess their biological function in calcium binding, MLC recombinant proteins were purified to analyze their calcium binding activity in vitro. Results: Two cDNA clones encoding MLC1 and MLC3 were identified from the fast white muscles of the spotted mandarin fish, Siniperca scherzeri. Sequence analysis revealed that MLC1 and MLC3 in the spotted mandarin were not produced by alternative splicing as reported in avians and rodents but were encoded by two different genes. MLC1 and MLC3 proteins contained two EF-hand domains, which are helix-loop-helix structural domains found in a large family of calcium-binding proteins. Conclusion: These two EMLC genes exhibited a muscle-specific expression. Moreover, the distinct patterns of expression of MLC1 and MLC3 had overlapped during the skeletal muscle development in the early embryonic stage. MLC1 and MLC3 recombinant proteins were able to bind Ca2+ in vitro, which is a typical characteristic of EF-hand superfamily proteins.