Immunological evidence that anemone repair proteins include replacement linkages

In response to damage to hair bundles caused by exposure to calcium free buffers, sea anemones secrete large protein complexes named 'repair proteins' that rapidly restore structural integrity and function to hair bundles. A specific chromatographic fraction of the repair protein mixture, named 'fraction beta', has biological activity comparable to the complete repair protein mixture (Watson et al., 1998, Hear. Res. 115, 119-128). In this study, we find that polyclonal antibodies raised against deglycosylated fraction beta specifically bind fraction beta on Western blots. Anti-fraction beta delays the normal recovery of vibration sensitivity in experimental animals (i.e., those with hair bundles damaged by calcium free buffers). Moreover, anti-fraction beta disrupts vibration sensitivity in control animals (i.e., those with healthy hair bundles). Experimentally damaged hair bundles subsequently exposed to repair protein and then processed for immunoelectron microscopy show labeled linkages interconnecting stereocilia of the hair bundle. Immunofluorescence microscopy confirms strong labeling of hair bundles treated with repair proteins and only weak labeling of tips of hair bundles from control animals. Immunofluorescence microscopy indicates stores of repair proteins in gland cells of the body column in control animals and in gland cells of the mouth in experimental animals. Repair biological activity is confirmed in column purified homogenates of these tissues. Apparently repair proteins are delivered to damaged hair bundles in mucus carried by beating cilia.