Molecular and functional characterization of a novel stefin analogue in large yellow croaker (Pseudosciaena crocea)

In this paper, we report the molecular cloning of a novel stefin analogue from the spleen of large yellow croaker Pseudosciaena crocea (Lycstefin). The open reading frame (ORF) of 297 nucleotides (nt) of Lycstefin encodes a protein of 99 amino acids (aa) with a putative molecular weight of 11kDa, in which no signal peptide and potential N-glycoslation site are predicted. The deduced Lycstefin possesses the structural features of the mammalian stefins, including two conserved motifs known to interact with the active sites of family C1 cysteine peptidases: one glycine in the N-terminal region (G(6)) and Gln-Xaa-Val-Xaa-Gly motif (Q(48)LVAG(52)). It shares 32-47.5% aa sequence identity to the sequences found in mammals and other fish species and is rich in cysteine residues (seven cysteines). Genomic analysis revealed that Lyccys gene, 757 nt long, consisted of three exons and two introns. The Lycstefin gene was constitutively expressed in various tissues examined although at different levels. Upon stimulation with poly(I:C) or inactivated trivalent bacterial vaccine, Lycstefin transcript was significantly up-regulated in spleen and head kidney while down-regulated in blood. Immuno-electron microscopy showed that Lycstefin was mainly localized in the cytoplasm of spleen cells of large yellow croaker, and also in the nucleus. Recombinant Lycstefin protein fused with glutathione S-transferase (rLycstefin) was shown to have strong inhibitory activity against papain with a K(i) of 1.3x10(-13)M. The in vivo experiments revealed that Lycstefin could not modulate the expression levels of large yellow croaker tumor necrosis factor-alpha2 (TNF-alpha2) and interleukin-10 in spleen and head kidney. To our knowledge, this is the first report on the molecular and functional identification of a stefin analogue in bony fish.