Yeast two-hybrid (Y2H) screens were used to test for interactions between leukocyte cell-derived chemotaxin 2 (LECT2) and a liver cDNA expression library of ayu, Plecoglossus altivelis. Of the 9 independent interacting clones identified, 2 were identical and closely related to C-type lectin receptor (CLR) genes of fish, while the other 7 were partial sequences from transferrin genes. Ayu CLR (aCLR) showed similarity to immune-relevant mammalian receptors in terms of amino acid sequence and overall organization within the C-type lectin-like domain (CTLD). The aCLR transcript was detected with the highest levels in the head kidney and peripheral blood leukocytes (PBLs), and more weakly in the heart, liver and gill. The interaction between aCLR and ayu LECT2 (aLECT2) was confirmed by in vitro co-immunoprecipitation of the two proteins. This interaction may be responsible for the "neutrophil-chemotactic" characteristic of LECT2. Y2H assays using different parts of the two proteins showed that the CTLD part of aCLR was involved in the interaction with mature aLECT2, and the contact structure of CTLD was essential for the interaction. The identification of this CLR/LECT2 interaction sheds light on the mechanism of serum LECT2 changes resulting in cellular immune responses.
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