Date of Graduation

2004

Document Type

Dissertation/Thesis

Abstract

This study demonstrated that rainbow trout muscle contains at least two forms of the Ca2+-dependent neutral protease calpain along with their endogenous inhibitor calpastatin (CAST). These calpain-system components can be separated with one DEAE-anion exchange column. Rainbow trout muscle calpains are less sensitive to Ca2+ than their mammalian counterparts. We described cloning and sequencing of the rainbow trout calpain small (regulatory) subunit (cpns) cDNA. This represents the first fish and lower vertebrate full cDNA of cpns. The rainbow trout cpns cDNA was used to retrieve the zebra fish and Japanese flounder homologues. We presented evidence that fish cpns unlike the conventional mammalian predominant isoform, cpns1, is lacking the glycine-rich region of Domain V. Because this region is known to play a role in membrane targeting, this divergent cpns suggests potentially different functional and activation mechanisms of the fish calpain system. A phylogenetic tree for the cpns gene super-family has been constructed and the evolution of cpns considered. In addition, we identified and described the molecular characterization of the rainbow trout calpain catalytic subunits Capn1 and Capn2. The cDNA sequence for Capn1 encodes a protein of 704 amino acids with a calculated molecular mass of 79.9 kDa. The amino acid sequence shows 66% and 86% identity with the mouse and zebrafish Capn1, respectively. The Capn2 cDNA codes for a protein consisting of 701 amino acid residues with a calculated molecular mass of 78.2 kDa. The protein shows 65% amino acid sequence identity with the mouse and chicken Capn2. The two isozymes of rainbow trout have the characteristic domains: D-I (pro peptide), D-II (cysteine catalytic site), D-III (electrostatic switch), and D-IV (contains five EF-hands). Furthermore, we identified the first fish CAST cDNAs. Different isoforms of the rainbow trout CASTs were described: Long (CAST-L), 2600-bp, 497 AA and short (CAST-S), with 2 splice variants, isoforms. CAST-S variant 1 (2132-bp) coded for 296 AA and variant 2 (2246-bp), with a premature stop codon, yielded 154 AA. Fish CASTs are novel in that they have the fewest number, one or two out of four, of the repetitive inhibitory domains reported thus far. Zebra fish CAST was retrieved from the GenBank database using trout CASTs. The rainbow trout: CAST-L showed 19 and 44%; CAST-S variant 2 showed 25 and 51% amino acid sequence identity with mouse and zebra fish CASTs, respectively. We also reported that the postmortem activation of calpains, by incubation in CaCl2, could accelerate fish muscle proteolysis and thereby affect texture development, particularly in early post harvest period. In addition, because starvation induces muscle wasting, we tested the hypothesis that starvation could affect regulation of the calpain system in muscle. Starvation of rainbow trout fingerlings (15-20 g) for 35 days stimulated the expression of Capn1, Capn2 (p<0.01), CAST short and long isoforms (p<0.05) as measured by real time RT-PCR. The net increase in the calpain/CAST ratio at the mRNA led to a 1.23 fold increase in the calpain catalytic activity. This suggests a potential role of calpains in protein mobilization as a source of energy under fasting condition. Furthermore, we measured the expression of calpain/CAST mRNA by the real time RT-PCR in three rainbow trout strains, differing in growth rate and fillet firmness, fed high energy or control diets. CAST-L and S expressions were significantly lower (p<0.01) in the strain which had the slowest growth rate, regardless of the feed type, and the softest fillets in fish fed the control diet, than the other two strains. These results enhance understanding of calpain proteolytic activity and its regulation in ante-mortem and postmortem fish muscle. The results also highlight the significance of the calpain system to the aquaculture industry. Questions can now be addressed regarding how this pathway is regulated in normal growth, what is its relationship to fish feeding efficiency and what is the role of calpains in fish muscle during refrigerated storage and their effects on fillet quality and functionality. Answers to these questions will assist in improving the aquaculture and seafood industries.

Download

Share

COinS