Molecular cloning, expression and characterization of myofibril-bound serine proteinase from yellow croaker (Larimichthys crocea)

Myofibril-bound serine proteinase (MBSP) is a serine proteinase which binds closely to myofibrillar proteins and is ubiquitous in animal muscle. MBSP is effective in the degradation of myofibrillar proteins, including myosin heavy chain (MHC), α-actinin, actin, and tropomyosin (TM) and is thus regarded as an important proteinase responsible for the metabolism of fish muscle in vivo, and is also considered as one of the main endogenous proteases causing the modori phenomenon. However, most research on fish MBSP focused on freshwater fish, and the research on MBSP from marine fish is still quite limited. In this study, the gene of MBSP from yellow croaker (Larimichthys crocea) was predicted by bioinformatics. Recombinant protein (Lc-rMBSP) was obtained by gene cloning and construction of pichia pastoris expression system. Enzymatic properties of Lc-rMBSP including molecular weight, substrate specificity, optimum temperature and pH were analyzed and its secondary structure was detected by circular dichroism. The three-dimensional structure of Lc-MBSP was analyzed by homology modeling. Sixteen trypsin-like genes in the genome of yellow croaker were identified. Phylogenetic tree analysis and multi-sequence alignment were performed between these genes and MBSPs in freshwater fish. The highest homology (57%) gene sequence (Lc-MBSP) was thus obtained, which had an open reading frame of 735 bp encoding 244 amino acid residues. The molecular weight of the recombinant protein Lc-rMBSP was~28 ku, and its optimal temperature and pH were 50 °C and 8.0, respectively. Circular dichroism analysis showed that temperature had a great influence on its secondary structure, and the thermal denaturation was irreversible. The thermal denaturation temperature of Lc-rMBSP was (62.82±1.09) °C, suggesting it is a thermally stable proteinase. Lc-rMBSP showed high hydrolytic activity toward MHC and TM in a temperature range of 40~60 °C. Lc-rMBSP hydrolyzed substrate Boc-Leu-Lys-Arg-MCA most effectively, and it specifically cleaved substrates containing arginine residues on the carboxyl side, which was similar to MBSP from Saurida undosquamis. The three-dimensional structure of Lc-MBSP was obtained by homology modeling. Its catalytic triplet was His-61, Asp-105 and Ser-198; its substrate binding pocket (Ser-192, Gly-215 and Ser-225) was slightly different from common trypsin-like proteinases, which was quite possibly the main reason for the difference in substrate specificity between Lc-rMBSP and other trypsins. Our present work provides a theoretical reference for the investigation of MBSP from marine fish.