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.
