Peroxinectin, a novel cell adhesion protein from crayfish blood.

From blood cells of the crayfish Pacifastacus leniusculus a 76-kDa protein that mediated attachment and spreading of the crayfish blood cells was purified. The cDNA for this cell adhesion protein was isolated, cloned, and sequenced. The deduced protein sequence was significantly similar to one family of peroxidases, e.g., myeloperoxidase. Consistently, the 76-kDa protein, for which we propose the name peroxinectin, had peroxidase activity. A synthetic peptide derived from the peroxinectin sequence containing Lys-Gly-Asp mimicked the cellular activity of the intact protein, implicating this sequence as the cell-binding site. Peroxinectin is the first cell adhesion molecule cloned from invertebrate blood and, to our knowledge, the first protein from any organism that combines being a cell adhesion ligand and a peroxidase.

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 myeloperoxidase), from the crayfish Pacifastacus leniusculus, was shown by immuno-fluorescence to bind to the surface of crayfish blood cells (haemocytes). In order to identify a cell surface receptor for peroxinectin, labelled peroxinectin was incubated with a blot of haemocyte membrane proteins. It was found to specifically bind two bands of 230 and 90 kDa; this binding was decreased in the presence of unlabelled peroxinectin. Purified 230/90 kDa complex also bound peroxinectin in the same assay. In addition, the 230 kDa band binds the crayfish beta-1,3-glucan-binding protein. The 230 kDa band could be reduced to 90 kDa, thus showing that the 230 kDa is a multimer of 90 kDa units. The peroxinectin-binding protein was cloned from a haemocyte cDNA library, using immuno-screening or polymerase chain reaction based on partial amino acid sequence of the purified protein. It has a signal sequence, a domain homologous to CuZn-containing superoxide dismutases, and a basic, proline-rich, C-terminal tail, but no membrane-spanning segment. In accordance, the 90 and 230 kDa bands had superoxide dismutase activity. Immuno-fluorescence of non-permeabilized haemocytes with affinity-purified antibodies confirmed that the crayfish CuZn-superoxide dismutase is localized at the cell surface; it could be released from the membrane with high salt. It was thus concluded that the peroxinectin-binding protein is an extracellular SOD (EC-SOD) and a peripheral membrane protein, presumably kept at the cell surface via 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 peroxinectin may mediate, or regulate, cell adhesion and phagocytosis; it may also be important for efficient localized production of microbicidal substances.

Identification and cloning of an integrin beta subunit from hemocytes of the freshwater crayfish Pacifastacus leniusculus.

We have cloned and sequenced a beta subunit of integrin from a cDNA library of crayfish hemocytes. This beta integrin shows great similarity to beta integrin subunits from other animals; the highest is towards beta pat-3 from Caenorhabditis elegans followed by beta PS from Drosophila melanogaster. By immunoblotting with antibodies raised towards a synthetic peptide corresponding to a part of the cytoplasmic region of the deduced protein sequence, it was shown that the integrin is present in the membrane of the hemocytes. This is the first integrin found in hemocytes of an invertebrate animal and this finding opens the door for further investigations on integrins and their role in the invertebrate immune system.