title: Molecular cloning and expression of ovarian cathepsin D in seabream, Sparus aurata.
abstract: In marine fish producing pelagic eggs, the acquirement of buoyancy by the eggs through the hydration process is a key event of reproduction; moreover, the yolk proteolysis, which leads to buoyancy, seems to affect the fertility and survival of the spawned eggs. Recently we demonstrated that cathepsin D is the aspartic protease responsible for this intraoocytic processing of vitellogenin into yolk proteins. In the present study, we isolated, cloned, and sequenced the cDNA encoding cathepsin D and studied expression of the message by Northern blotting and whole-mount in situ hybridization. The full-length seabream cathepsin D cDNA is 1837 base pairs long, encoding a protein of 400 amino acids (aa) consisting of a signal peptide of 19 aa, a prosequence of 44 aa, and a mature peptide of 336 aa. An absolute sequence conservation at the aspartyl residues (+33 and +221) was found, and there are three potential N-glycosylation sites at +70, aa +189, and aa +274. The aa sequence of seabream cathepsin D reveals a high degree of sequence similarity with cathepsin D mRNAs from other organisms (73% sequence homology to mouse and rat, 72% to human and trout, 69% to chicken, 66% to pig, and 65% to Xenopus). The cathepsin D mRNA in floating eggs was present as a single band that was approximately 1.9 kilobases (kb) in size, while in the sinking eggs there were several fast-migrating bands (size range 1.3-0.2 kb). Whole-mount hybridization was used to investigate transcription of cathepsin D in the developing embryo; during the hatching period, cathepsin D mRNA-positive cells were distributed in a wide region between the trunk and the tail, and in the ventral region over the yolk ball. The highest levels of cathepsin D enzymatic activity were found in the sinking eggs and during the hatching period of embryonic development. These data suggest that cathepsin D can be considered a possible marker for egg quality.
authors.affiliation: Dipartimento di Scienze Morfologiche e Biochimiche Comparate, Università di Camerino, 62032 Camerino, Italy. o.carnevali@cambio.unicam.it
authors.forename: O
authors.initials: O
authors.lastname: Carnevali
authors.forename: F
authors.initials: F
authors.lastname: Centonze
authors.forename: S
authors.initials: S
authors.lastname: Brooks
authors.forename: I
authors.initials: I
authors.lastname: Marota
authors.forename: J P
authors.initials: JP
authors.lastname: Sumpter
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002402:Cathepsin D / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D005314:Embryonic and Fetal Development; D005260:Female; D005333:Fetus / Q000201:enzymology; D015870:Gene Expression; D005865:Gestational Age; D006031:Glycosylation; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010063:Ovum / Q000201:enzymology; D010473:Perciformes / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D002402:Cathepsin D