Molecular characterisation of ovarian cathepsin D in the rainbow trout, Oncorhynchus mykiss.
In fish, cathepsin D, an aspartyl protease, is believed to mediate the processing of yolk proteins in the oocyte. Cathepsin D, therefore, is vital for the production of a viable egg. This study set out to isolate and sequence the cDNA encoding cathepsin D, and to determine the developmental expression of the message in the ovary and subsequently during embryogenesis in the rainbow trout, Oncorhynchus mykiss. The full-length trout cathepsin D cDNA is 1847 base pairs (bp) long, encoding a protein of 400 amino acids (aa). The sequence consists of a putative signal peptide of 18 aa, a prosequence extending 46 aa and a mature peptide of 336 aa. The deduced sequence of rainbow trout ovarian cathepsin D shows significant homology with cathepsin D in mammals (human; 81% aa similarity), in the chicken (80% aa similarity) and in Xenopus (74% aa similarity). Our data support the contention that the primary structure of cathepsin D is highly conserved across the vertebrate phyla, from mammals to fish. Unlike cathepsin Ds in other species, however, rainbow trout cathepsin D appears to have only one putative N-glycosylation site, rather than two. The mRNA for 'ovarian' cathepsin D was expressed in both ovarian and non-ovarian tissues (liver, muscle, spleen and testis). During the development of the ovary, the highest expression levels of cathepsin D mRNA were seen at around the onset of vitellogenesis, a time when the oocytes are starting to sequester large quantities of yolk proteins. Northern hybridisation did not detect cathepsin D mRNA in either unfertilised eggs, or in fertilised eggs until after gastrulation, indicating that there is little, if any, de novo synthesis of this message at these stages of development. However, the mRNA for cathepsin D was detectable at the eyed embryo stage, and the expression of the gene increased towards the end of embryonic development.