Cloning and characterization of integrin alpha subunits from the solitary ascidian, Halocynthia roretzi

Recent molecular and biochemical analysis has revealed the presence of an o psonic complement system in the solitary ascidian, Halocynthia roretzi, com posed of at least C3, two mannan binding protein-associated serine protease s, and factor B, To elucidate further the structure and function of this ap parently primitive complement system in the urochordates, we looked for the ascidian complement receptor type 3 (CR3), or type 4 (CR4), which are memb ers of the leukocyte integrin family in mammals. Using degenerate primers, we isolated two integrin a subunits (alpha (Hr1) and alpha (Hr2)) from the hemocyte mRNA of H. roretzi, by RT-PCR, and the entire coding sequence of a lpha (Hr1) was determined from cDNA clones. alpha (Hr1) contains an I domai n, the inserted domain characteristic of a subset of mammalian alpha subuni ts, including the leukocyte integrin family, A phylogenetic tree constructe d for the alpha subunits also supports the ancestral position of alpha (Hr1 ) in the monophyletic cluster of I domain-containing alpha integrins, The a lpha (Hr1) gene shows hemocyte-specific expression on Northern blot analysi s. Western blot analysis and immunocytochemical staining of the hemocytes o f H. roretzi using anti-alpha (Hr1) Ab showed that alpha (Hr1) subunits exi st on the surface of a subpopulation of phagocytic hemocytes, Furthermore, anti-alpha (Hr1) Ab inhibited C3-dependent phagocytosis, but not basic phag ocytosis, of yeast cells by ascidian hemocytes, These observations strongly suggest that alpha (Hr1) constitutes an integrin molecule on the hemocytes of H. roretzi that functions as an ancestral form of CR3 and CR4 and media tes phagocytosis in the primitive complement system of the ascidian.