Characterization of a pattern recognition protein, a masquerade-like protein, in the freshwater crayfish Pacifastacus leniusculus

A multifunctional masquerade-like protein has been isolated, purified, and characterized from hemocytes of the freshwater crayfish, Pacifastacus leniu sculus. It was isolated by its Escherichia coli binding property, and it bi nds to formaldehyde-treated Gram-negative bacteria as well as to yeast, Sac charomyces cerevisiae, whereas it does not bind to formaldehyde-fixed Gramp ositive bacteria. The intact masquerade (mas)-like protein is present in cr ayfish hemocytes as a heterodimer composed of two subunits with molecular m asses of 134 and 129 kDa. Under reducing conditions the molecular masses of the intact proteins are not changed. After binding to bacteria or yeast ce ll walls, the mas-like protein is processed by a proteolytic enzyme. The 13 4 kDa of the processed protein yields four subunits of 65, 47, 33, and 29 k Da, and the 129-kDa protein results in four subunits of 63, 47, 33, and 29 kDa in 10% SDS-PAGE under reducing conditions. The 33-kDa protein could be purified by immumoaffinity chromatography using an Ab to the C-terminal par t of the mas-like protein. This subunit of the mas-like protein has cell ad hesion activity, whereas the two intact proteins, 134 and 129 kDa, have bin ding activity to LPSs, glucans, Gram-negative bacteria, and yeast. E. coli coated with the mas-like protein were more rapidly cleared in crayfish than only E. coli, suggesting this protein is an opsonin. Therefore, the cell a dhesion and opsonic activities of the mas-like protein suggest that it play s a role as an innate immune protein.