Binding specificity and recognition roles of the Atlantic salmon serum C-type lectin.

摘要

Lectins are proteins that bind to sugars. In animals, there is a wide variety of lectins and most of these proteins have roles in immunity. This project focused on a soluble Ca2+-dependent (C-type) lectin isolated from the serum of Atlantic salmon (Salmo salar). Three heterologous cell systems were evaluated for their ability to express salmon serum lectin (SSL). Although the SSL was not successfully expressed in Pichia pastoris, expression in Escherichia coli and insect Drosophila melangolaster S2 cells resulted in low levels of correctly processed and functional SSL. Substantial quantities of pure native lectin were obtained by implementing an efficient purification procedure consisting of whole-yeast-affinity and mannan-affinity chromatography. To determine the immunological roles of this lectin in salmon, its interaction with the bacterial surface was investigated. SSL was shown to bind to important bacterial pathogens and yeast including, Pseudomonas fluorescens, Aeromonas salmonicida, A. hydrophila, P. pastoris, and Saccharomyces cerevisiae, but not to Yersinia ruckeri. In antimicrobial assays, SSL showed no activity against E. coli, Bacillus subtilis, S. cerevisiae, and A. salmonicida, but it was found to agglutinate E. coli. The binding of SSL to the two major surface molecules of A. salmonicida, lipopolysaccharide (LPS) and A-layer protein was investigated by western blotting and enzyme-linked immunosorbent assays, which showed mannose-inhibitable binding to LPS. Binding of labelled SSL to a glycan array performed by the Consortium for Functional Glycomics demonstrated preferred binding to glycans with terminal N-acetylglucosamine. These findings suggest that SSL has ligand specificity that is rather precise at the molecular level, but that allows recognition of a wide variety of pathogens.