Characterization and expression of an EB1 protein in Ictalurus punctatus neutrophils

We have identified an EB1 gene (CfEB1) and protein in channel catfish neutrophils. The complete cDNA sequence is 1725 bp and the putative protein is composed of 258 amino acids. Western blot analysis of channel catfish neutrophil cell membrane with a monoclonal antibody (14I) yielded a 28 kD protein band with the protein preparation. Aside from neutrophils only a small percentage of other cells tested expressed detectable amounts of EB1 as determined by flow cytometry. Furthermore, EB1 expression increased after phorbol dibutyrate stimulation of neutrophils or incubation of catfish neutrophils with Edwardsiella ictaluri. Nonpermeabilized, fixed catfish neutrophils demonstrated immunofluorescent staining with 14I indicating that EB1 is apparently externally oriented in catfish neutrophils. This is the first report of the external orientation of the EB1 molecule. Because of its increase after stimulation and detection on cell membranes, EB1 may participate in catfish neutrophil cell regulation, signal transduction, or cell membrane changes necessary for phagocytosis.