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

The cDNA of a cystatin analogue was isolated from the spleen Smart cDNA library of large yellow croaker Pseudosciaena crocea (Lyccys). The open reading frame (ORF) of 354 nucleotides (nt) of Lyccys encodes a protein of 118 amino acids (aa), containing a 21-aa signal peptide and a 97-aa mature polypeptide. The deduced Lyccys possessed structural features of the Family II cystatins, including three evolutionally conserved motifs known to interact with the active sites of cysteine peptidases: Gly at the N-terminus (Gly(25)), Gln-X-Val-X-Gly motif (Q(69)LVAG(73)) and Pro-Try pair at the C-terminus (P(106)W(107)). Genomic analysis revealed that Lyccys gene, spanning 2297 nt, consisted of three exons and two introns. The Lyccys gene was constitutively expressed in all eight tissues examined although at different levels. Real-time PCR analysis revealed that Lyccys transcript in spleen and kidney was obviously up-regulated by poly(I:C) or inactivated trivalent bacterial vaccine, while in blood its expression was down-regulated. Immuno-electron microscopy showed that Lyccys was mainly localized to the rough endoplasmic reticulum (rER) or in the vesicular structures in spleen and kidney cells. Recombinant Lyccys protein fused with glutathione S-transferase (rLyccys) was shown to have remarkable protease-inhibitory activity and well affinity binding to papain (with a K(i) of 1.3x10(-13) M). An in vivo administration of rLyccys could significantly up-regulate the expression levels of large yellow croaker tumor necrosis factor-alpha2 (TNF-alpha2) and interleukin-10 in spleen and kidney, but to a lesser extent increase TNF-alpha1 expression. These results suggest that the Lyccys is a secreted inhibitor of cysteine proteinases, which may have an immunomodulatory function in inflammation response.