Cloning and sequencing of caspase 6 in rainbow trout, Oncorhynchus mykiss,and analysis of its expression under conditions known to induce apoptosis

The rainbow trout caspase 6 gene has been cloned and sequenced. The open re ading frame consisted of 906 bp, which translated into a protein of 302 ami no acids, containing the caspase active site pentapeptide (QACRG) and the c aspase family signature (HADADCFVCVFLSHG). Amino acids involved in catalysi s and those known to form the P1 carbohydrate binding pocket were conserved . Phylogenetic tree analysis showed a tight grouping with other known caspa se 6 genes. Conserved aspartic acid residues at positions 33, 191 and 202 s uggested that this molecule is produced as a proenzyme that is subsequently cleaved to release active subunits, with the region between Asp-191 and Al a-203 acting as a linker that is cleaved out. RT-PCR analysis revealed that the trout caspase 6 gene was expressed in brain, blood, gill, liver, head kidney and spleen. Addition of LPS or cortisol to head kidney leucocyte cul tures had no effect upon caspase 6 expression. However, addition of LPS aft er preincubation with cortisol increased expression relative to control cul tures. Incubation with RU486 abrogated this effect, confirming it was media ted via glucocorticoid receptors. Lastly, a confinement stress in vivo incr eased caspase 6 expression. The data are discussed with respect to the immu noregulatory role of apoptosis in fish immune responses. (C) 2001 Elsevier Science Ltd. All rights reserved.