Characterization of novel ascidian β integrins as primitive complement receptor subunits

Integrin-type complement receptors play pivotal roles in the effector mechanisms of the complement system. Previously, we identified an integrin alpha subunit, alpha(Hr1), from the solitary ascidian, Halocynthia roretzi, which is involved in the complement-dependent phagocytic activities of ascidian hemocytes. To identify integrin beta subunits that pair with alpha(Hr1) to compose ascidian complement receptors, genes encoding beta subunits were cloned and characterized for their binding property to alpha(Hr1). Using degenerate primers and RT-PCR, two integrin beta transcripts (beta(Hr1) and beta(Hr2)) were isolated from H. roretzi hemocyte total RNA and the entire coding sequences of both cDNA species were determined. The putative primary structure of each ascidian gene product retained domains characteristic for integrin beta subunits. Phylogenetic analysis revealed that beta(Hr1) and beta(Hr2) are located outside of vertebrate integrin beta groups, comprising an independent cluster specific for the ascidian lineage. The alpha(Hr1), beta(Hr1) and beta(Hr2) subunits all showed hemocyte-specific expression on Northern blot analysis, and recombinant proteins of both beta subunits could bind to alpha(Hr1) on insect cells. The beta(Hr1) subunit was expressed especially on the surface of ascidian phagocytic hemocytes, such as phago-amoebocytes. In the immunoprecipitation analysis of ascidian hemocytes using anti-beta(Hr1) antiserum, alpha(Hr1) was coprecipitated with beta(Hr1). These observations showed that beta(Hr1), and possibly beta(Hr2) too, binds to alpha(Hr1) to comprise integrin molecules on ascidian hemocytes, which act as ancestral forms of complement receptors in the primitive complement system of ascidians.