Expression And Function Analysis Of A Novel Gene Of The Ly6 Superfamily In Zebrafish

Ly6/uPAR/CD59/netuotoxin superfamilies ?Ly6SF? called snake toxin family present in many metazoans. Ly6SF possess conserved LU domain containing eight to ten conserved cysteine residues which form 4 to 5 conserved disulfide bonds. The disulfide bonds form so-called'three-fingered protein'domains ?TFPDs?. Many members of Ly6 superfamily are glycosylphosphatidylinositol ?GPI?-anchored cell surface proteins. In human, mouse and drosophila, the function and expression of some Ly6 genes have been documented. They are found to exert a wide variety of biological effects. For example, human GPI-anchored CD59 not only protects host cells from complement-induced lysis but also binds to CD2, transducing activation signals within T cells. Murine Lynx protein dampens the activity of cholinergic system through direct interaction with nicotinic receptors. And the fruitfly protein encoded by Boudin ?bou? is essential for tracheal morphogenesis in the embryo and contributes to the maintenance of the paracellular barrier and the organisation of the septate junctions. Members of Ly6 superfamily, like the secreted Ly6/urokinase-type plasminogen activator receptor ?uPAR?-related protein-1 ?SLURP 1? which is highly expressed in human cornea, are also present in secreted forms. Despite the extensive studies on the members of Ly6 superfamily, functional identification of Ly6 family genes remains scarce.The zebrafish ?Danio rerio?, is an important model organism in scientific research. We have identified 28 uncharacterized LU domain-containing genes in different chromosomes of zebrafish by the multiple sequence alignment to identify similarities and homolog. Among them, two genes are found to be expressed in the lateral line neuromast in a tissue-specific manner. One gene expresses in the ions of epidermal cells. Moreover, another gene expresses in the epiphysis ?XP₆98291?. The different expression pattern indicate that Ly6 superfamily members in zebrafish execute a variety of functions.Here we identified a novel Ly6 gene located on the chromosome 15 in zebrafish?XP₀03200124?, and named it Lypc highlighting its predominant expression in the pigment cells. Both homology modeling and sequence comparison revealed that Lypc has typical features of Ly6 family members. It encodes a 124 amino acid protein, which contains a signal peptide of 20 amino acids in N-terminus and a mature peptide of 104 amino acids. The predicted protein shows a set of 10 conserved cysteine residues forming 5 disulfide bonds. As members of Ly6 superfamily is not well conserved, it is thus difficult to determine any orthologous genes in other species only by bioinformatic analysis. Whole mount in situ hybridization showed that Lypc was expressed in the trunk pigment cells and retinal pigment cells. To pinpoint the pigment cell type in which Lypc is expressed in the trunk, we investigated the expression pattern of Lypc in the pigment cells of mutant Casper, which is a Roy, Nacre double homozygous mutant with a transparent body due to loss of melanocytes and iridopore from 1-cell to adult and normal retinal pigment cells.It was found that the expression of Lypc in the trunk disappeared in Casper mutant compared to wild type. This further proved that Lypc is indeed expressed in the pigment cells, mostly in the melanocyte and iridophore.Moreover, real-time quantitative PCR demonstrated that the expression of Lypc was subjected to diurnal variation regulation, i.e. the expression of Lypc displays a clear rhythmic pattern, and dark exposure apparently stimulated its expression.Collectively, these data indicate that Lypc is a novel dark-inducible Ly6 member with a predominant expression in the pigment cells of zebrafish, laying a foundation for further elucidation of its functions.