Characterisation of capn1, capn2-like, capn3 and capn11 genes in Atlantic halibut (Hippoglossus hippoglossus L.): Transcriptional regulation across tissues and in skeletal muscle at distinct nutritional states.

The typical calpain proteases are a subset of a wider superfamily and regulate a broad spectrum of physiological processes. Here we characterised Atlantic halibut complete-coding orthologues of calpain-1, calpain-2-like, "muscle-specific" calpain-3, plus calpain-11, a recently recognised vertebrate-wide family member. Phylogenetic analysis established the relationship of each sequence within a comprehensive framework of vertebrate calpains, including teleost paralogues. This approach provided significant insight into the evolution of teleost calpains. For example, teleost sequences considered calpain-2 orthologues formed a monophyletic clade external to sister clades for tetrapod calpain-2 and vertebrate calpain-8. Thus, teleost "calpain-2" is likely not directly orthologous to tetrapod calpain-2 and represents a calpain-2-like protein. The characteristic domain structure of typical calpains was observed in each halibut sequence, although calpain-3, as for other teleosts, retained only one (IS2) of three further domains found in human calpain-3 (NS, IS1 and IS2). Transcripts for capn1, capn2-like and capn11 were widely detected across eleven halibut tissues, whereas capn3 was detected in striated muscles, spleen and ovary, but absent or relatively less abundant in other tissues. We assessed the transcript expression of each calpain gene in fast-twitch skeletal muscle where nutritional state was altered with 60days feed restriction, followed by 60days satiation refeeding. Measured by quantitative real-time PCR, capn1 transcript levels were highest during maximal fasting and then steadily decreased with refeeding, where muscle was in net positive protein balance. Conversely capn2-like showed little response, whereas capn3 and capn11 transcript levels were lowest at maximal fasting before being strongly constitutively upregulated with subsequent refeeding. Halibut capn3 transcript abundance was on average 6.5, 23.7 and 5.9 fold greater than capn1, capn2-like and capn11 respectively in skeletal muscle across nutritional states. In turn, transcript levels of capn1 and capn11 were invariably higher than capn2-like, but were dependent on nutritional state compared to each other. The differential regulation of these genes in response to nutritional status suggests distinct roles for typical calpain family members in regulating the balance between catabolism and growth in teleost skeletal muscle.

Functional significance of the shark Na,K-ATPase N-terminal domain. Is the structurally variable N-Terminus involved in tissue-specific regulation by FXYD proteins?

The proteolytic profile after mild controlled trypsin cleavage of shark rectal gland Na,K-ATPase was characterized and compared to that of pig kidney Na,K-ATPase, and conditions for achieving N-terminal cleavage of the alpha-subunit at the T(2) trypsin cleavage site were established. Using such conditions, the shark enzyme N-terminus was much more susceptible to proteolysis than the pig enzyme. Nevertheless, the maximum hydrolytic activity was almost unaffected for the shark enzyme, whereas it was significantly decreased for the pig kidney enzyme. The apparent ATP affinity was unchanged for shark but increased for pig enzyme after N-terminal truncation. The main common effect following N-terminal truncation of shark and pig Na,K-ATPase is a shift in the E(1)-E(2) conformational equilibrium toward E(1). The phosphorylation and the main rate-limiting E(2) --> E(1) step are both accelerated after N-terminal truncation of the shark enzyme, but decreased significantly in the pig kidney enzyme. Some of the kinetic differences, like the acceleration of the phosphorylation reaction, following N-terminal truncation of the two preparations may be due to the fact that under the conditions used for N-terminal truncation, the C-terminal domain of the FXYD regulatory protein of the shark enzyme, PLMS or FXYD10, was also cleaved, whereas the gamma or FXYD2 of the pig enzyme was not. In the shark enzyme, N-terminal truncation of the alpha-subunit abolished association of exogenous PLMS with the alpha-subunit and the functional interactions were abrogated. Moreover, PKC phosphorylation of the preparation, which relieves PLMS inhibition of Na,K-ATPase activity, exposed the N-terminal trypsin cleavage site. It is suggested that PLMS interacts functionally with the N-terminus of the shark Na,K-ATPase to control the E(1)-E(2) conformational transition of the enzyme and that such interactions may be controlled by regulatory protein kinase phosphorylation of the N-terminus. Such interactions are likely in shark enzyme where PLMS has been demonstrated by cross-linking to associate with the Na,K-ATPase A-domain.

Sequence, circulating levels, and expression of C-type natriuretic peptide in a euryhaline elasmobranch, Carcharhinus leucas.

The present study has examined expression and circulating levels of C-type natriuretic peptide (CNP) in the euryhaline bull shark, Carcharhinus leucas. Complementary DNA and deduced amino acid sequence for CNP in C. leucas were determined by RACE methods. Homology of CNP amino acid sequence in C. leucas was high both for proCNP and for mature CNP when compared with previously identified elasmobranch CNPs. Mature CNP sequence in C. leucas was identical to that in Triakis scyllia and Scyliorhinus canicula. Levels of expression of CNP mRNA were significantly decreased in the atrium but did not change in either the brain or ventricle following acclimation to a SW environment. However, circulating levels of CNP significantly increased from 86.0+/-7.9 fmol ml(-1) in FW to 144.9+/-19.5 fmol ml(-1) in SW. The results presented demonstrate that changes in environmental salinity influences both synthesis of CNP from the heart and also circulating levels in C. leucas. Potential stimulus for release and modes of action are discussed.

Regulation of Na,K-ATPase by PLMS, the phospholemman-like protein from shark: molecular cloning, sequence, expression, cellular distribution, and functional effects of PLMS.

In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Here we describe the complete coding sequence, expression, and cellular localization of PLMS in the rectal gland of the shark Squalus acanthias. The mature protein contained 74 amino acids, including the N-terminal FXYD motif and a C-terminal protein kinase multisite phosphorylation motif. The sequence is preceded by a 20 amino acid candidate cleavable signal sequence. Immunogold labeling of the Na,K-ATPase alpha-subunit and PLMS showed the presence of alpha and PLMS in the basolateral membranes of the rectal gland cells and suggested their partial colocalization. Furthermore, through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be specifically cleaved. Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. Both the E1 approximately P --> E2-P reaction, which is partially rate-limiting in shark, and the K+ deocclusion reaction, E2(K) --> E1, are accelerated. The latter may explain the finding that the apparent Na+ affinity was increased by the specific C-terminal PLMS truncation. Thus, these data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase alpha-subunit is important for the modulation of shark Na,K-ATPase activity.