| Lymphocyte antigen-6 (Ly-6) gene products were initially identified in mouse in 1970s. Ly-6 superfamily proteins are characterized by a Ly-6 (LU) domain that it is about 70 to 100 amino acids long and contains 8 to 10 cysteine residues that form a defined disulfide-bonding pattern, and termed the three-finger fold, Thus, they were also known as Three Finger Proteins. To date, a large number of members of the Ly-6 family were identified among many species and they are divided into two subfamilies: one subfamily includes secreted proteins such as SLURP1, SLURP2, SP10, PATE family or nonmammalian secreted cytotoxins, and the other one comprises GPI-anchored membrane proteins such as CD59, PSCA (prostate stem cell antigen), LYNX1 (Ly-6/neurotoxin 1).A wide range of Ly-6 superfamily members have been identified to date in a variety of species ranging from invertebrates to mammals. The members of Ly-6 superfamily showed diversified but specific expression pattern, consistant with that, they participated in different biological contexts including cell activity, migration and adhesion, regulation of neural excitability and immunoregulatory. In recent years, most the identified members of the family of Ly-6 are GPI anchored membrane proteins. The discovery and identification of secreted proteins are relatively few. Zebrafish has emerged as a popular vertebrate model because both genetic and experimental embryological methods can be well developed in this animal. In addition, few studies were reported to investigate the function of Ly-6 family in early development of zebrafish besides some general bioinformatics.In this study, we first report the identification and characterization of the secreted Ly2.1-3 proteins on the chromosome 2 in zebrafish and determine the expression pattern. Ly2.1-3 possess a conserved LU domain containing 10 position-specific conserved cysteines. In addition, they share similar three-finger structure with human CD59, SLURP 1 and other Ly-6 family members. According to the results of WISH and Real Time-PCR, In the early embryo, they all have different levels of maternal expression, then Ly2.1-3 were found to be expressed abundantly in endoderm, but they exhibited different expression pattern. Ly2.1-3 expressed in swim bladder and pneumatic duct. Ly2.2 possesses a specific expression in pancreas after 72hpf. Ly2.3 expressed throughout the digestive tract and in liver as well.It is well known that the digestive system plays a key role in innate immune and adaptive immune to defense against pathogens. The analysis with Antimicrobial Peptide Calculator and Predictor suggest that Ly2.1-3 possess cationic nature (pI=8.5 for Ly2.1, pI=8.2 for Ly2.2,pI= 8.2 for Ly2.3), high cysteine content with Hydrophobic ratios 46%,48%,47%. They might be expected to exhibit potent antimicrobial peptides (AMPs) activity. In this case, we investigate their possible roles during infection or under conditions that mimic an infection to counter pathogen attack via endotoxin, LPS challenge. Real time quantitative PCR results showed that, Ly2.1-3 expression levels have increased with injection of LPS in different stages of zebrafish embryos. These changes of Ly2.1-3 expression indicated that they may participate in the innate immune responses of the digestive system during endotoxin challenge in zebrafish early embryos/larvae.In summary, this paper identified three members of the Ly-6 superfamily on the chromosome 2 of zebrafish, Ly2.1, Ly2.2, Ly2.3, they are evolved by chromosomal gene duplication and all expressed in endoderm but exhibit different expression pattern,which suggest that they may perform different functions after duplication. They are upregulated by LPS injection and may participate in immune regulation in zebrafish digestive system in early embryonic development. |
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