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 reading frame consisted of 906bp, which translated into a protein of 302 amino acids, containing the caspase active site pentapeptide (QACRG) and the caspase family signature (HADADCFVCVFLSHG). Amino acids involved in catalysis and those known to form the P1 carbohydrate binding pocket were conserved. Phylogenetic tree analysis showed a tight grouping with other known caspase 6 genes. Conserved aspartic acid residues at positions 33, 191 and 202 suggested that this molecule is produced as a proenzyme that is subsequently cleaved to release active subunits, with the region between Asp-191 and Ala-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 cultures had no effect upon caspase 6 expression. However, addition of LPS after preincubation with cortisol increased expression relative to control cultures. Incubation with RU486 abrogated this effect, confirming it was mediated via glucocorticoid receptors. Lastly, a confinement stress in vivo increased caspase 6 expression. The data are discussed with respect to the immunoregulatory role of apoptosis in fish immune responses.
Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK.
K J
KJ
Laing
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J
Holland
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Bonilla
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Cunningham
C J
CJ
Secombes
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D001665:Binding Sites; D053178:Caspase 6; D020169:Caspases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D002645:Chickens; D016384:Consensus Sequence; D004790:Enzyme Induction / Q000187:drug effects; D019143:Evolution, Molecular; D005796:Genes; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D007962:Leukocytes / Q000166:cytology / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D015735:Mifepristone / Q000494:pharmacology; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000235:genetics* / Q000378:metabolism; D009928:Organ Specificity; D010802:Phylogeny; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013312:Stress, Physiological / Q000201:enzymology / Q000235:genetics
D006727:Hormone Antagonists; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D015735:Mifepristone; C505514:CASP6 protein, human; C505516:Casp6 protein, mouse; C505518:Casp6 protein, rat; D053178:Caspase 6; D020169:Caspases; D006854:Hydrocortisone