Identification Of The Proteins Interacting With Leukocyte Cell-derived Chemotaxin 2 Of Ayu, Plecoglossus Altivelis By Yeast Two-hybrid Screens

Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional protein, involved in cell growth, differentiation, damage/repair process and auto immune response. Recent studies indicated that LECT2 might participate in immune regulation of fish, but the role of LECT2 in vivo is unclear. To further elucidate the roles of LECT2 in the fish, a yeast two-hybrid system (Y2H) was used to screen an ayu liver cDNA library for proteins directly interacting with LECT2.In the present study, the nucleotide sequence of full length cDNA clone of ayu LECT2 (aLECT2) gene was determined. The aLECT2 was 547 nucleotide long excluding the poly(A) tail. The ORF was predicted to produce a protein of 156 amino acids with MW 17.0 kDa. Using expressed aLECT2 (FM253748) as bait in Y2H screens, 9 independent interacting clones were identified after two separate screenings of an ayu cDNA expression library (2.5×106 independent clones). Sequencing showed that 7 of the 9 clones were partial sequences from a Transferrin (aTf) gene, while the other two were sequences from a c-type lectin receptor (aCLR) gene.The complete cDNA sequence of aTf gene (FM875933) was 2246 nucleotide long excluding the poly (A) tail. The ORF was predicted to produce a protein of 693 amino acids with MW 75.9 kDa. Computer analysis revealed that the segment aTf185–289, which contained two iron binding residues, Tyr197 and His253, was located at the N-terminus of aTf N-lobe. It showed that aLECT2 was still interacted with the entire aTf in vivo in Saccharomyces cerecisiae. Y2H assays using different parts of the two proteins showed that the segment aTf185–289 was not involved in the interaction with mature aLECT2, while the transit peptide of aLECT2 couldn't interact with entire aTf. Y2H assays were used to examine the interactions between LECT2 and Tf in other animals. Just as in large yellow croaker and mouse, strong interactions were observed between LECT2 and Tf from the same species, but not from different species. According to reports in the literature, the interaction between LECT2 and Tf might be associated with the immune response to pathogenic infection.The complete cDNA sequence of aCLR gene (FN396582) was determined. It was 1265 nucleotides in length excluding the poly (A) tail. The ORF was predicted to produce a protein of 256 amino acids with MW 29.30 kDa. From the amino acid sequence analysis, ayu C-type lectin receptor was most similar to the previously reported Atlantic salmon (Salmo salar) C-type lectin receptor A (sCLRA). A plasmid pGADT7-aCLR with the complete aCLR ORF in a pGADT7 vector was constructed subsequently and retransformed back to yeast either alone or in combination with pGBKT7-aLECT2. It was comformed that aCLR interacted independently with aLECT2. Y2H assays using different parts of the two proteins showed that the CTLD part of aCLR was involved in the interaction with mature aLECT2. Mammalian DC-SIGN contains the most closely related complete CTLD to that of fish CLRs. The strong interaction between mouse DC-SIGN and mouse LECT2 were also detected by Y2H, indicating that serum LECT2 might affect DC-SIGN on the cell membrane. In conclusion, CLRs may be an important component involved in LECT2 signaling transduction, and CLR/LECT2 interaction may be responsible for the"neutrophil-chemotactic"characteristic of LECT2.