A novel membrane glycoprotein involved in ascidian hemocyte aggregation and phagocytosis

Invertebrate hemocytes undergo several cellular defense reactions. To clarify the molecular mechanisms for cellular recognition between hemocytes and also between hemocytes and foreign materials, we established hybridoma clones producing monoclonal antibodies that inhibit hemocyte aggregation (i.e. a cellular reaction between hemocytes) in the solitary ascidian, Halocynthia roretzi. The antibody, A74, also inhibited phagocytosis of foreign materials by H. roretzi hemocytes. Immunocytochemistry of H. roretzi hemocytes using A74 antibody revealed the localization of the A74 antigen on the surface of hemocytes. The A74 antigen, which is referred to as A74 protein, was purified from a hemocyte membrane preparation by three chromatographies on phenyl-Sepharose, A74 antibody-immobilized Sepharose and Mono Q. The A74 protein was a glycoprotein with a molecular mass of 160 kDa; N-glycosidase or neuraminidase treatment resulted in a reduction of its molecular mass. The N-terminal amino acid sequence of A74 protein showed little similarity to other known proteins. Thus, the A74 protein is a novel membrane protein that plays an important role in ascidian cellular defense reactions.