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.