A cell-surface superoxide dismutase is a binding protein for peroxinectin,a cell-adhesive peroxidase in crayfish

Peroxinectin, a cell-adhesive peroxidase (homologous to human myeloperoxida se), from the crayfish Pacifastacus leniusculus, was shown by immune-fluore scence to bind to the surface of crayfish blood cells (haemocytes). In orde r to identify a cell surface receptor for peroxinectin, labelled peroxinect in was incubated with a blot of haemocyte membrane proteins. It was found t o specifically bind two bands of 230 and 90 kDa; this binding was decreased in the presence of unlabelled peroxinectin, Purified 230/90 kDa complex al so bound peroxinectin in the same assay. In addition, the 230 kDa band bind s the crayfish beta-1,3-glucanbinding protein, The 230 kDa band could be re duced to 90 kDa, thus showing that the 230 kDa is a multimer of 90 kDa unit s. The peroxinectin-binding protein was cloned from a haemocyte cDNA library, using immune-screening or polymerase chain reaction based on partial amino acid sequence of the purified protein. It has a signal sequence, a domain h omologous to CuZn-containing superoxide dismutases, and a basic, proline-ri ch, C-terminal tail, but no membrane-spanning segment. In accordance, the 9 0 and 230 kDa bands had superoxide dismutase activity. Immune-fluorescence of non-permeabilized haemocytes with affinity-purified antibodies confirmed that the crayfish CuZn-superoxide dismutase is localized at the cell surfa ce; it could be released from the membrane with high salt. It was thus conc luded that the peroxinectin-binding protein is an extracellular SOD (EC-SOD ) and a peripheral membrane protein, presumably kept at the cell surface vi a ionic interaction with its C-terminal region. This interaction with a peroxidase seems to be a novel function for an SOD, The binding of the cell surface SOD to the cell-adhesive/opsonic peroxinec tin may mediate, or regulate, cell adhesion and phagocytosis; it may also b e important for efficient localized production of microbicidal substances.