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

Recent molecular and biochemical analysis has revealed the presence of an opsonic complement system in the solitary ascidian, Halocynthia roretzi, composed of at least C3, two mannan binding protein-associated serine proteases, and factor B, To elucidate further the structure and function of this apparently primitive complement system in the urochordates, we looked for the ascidian complement receptor type 3 (CR3), or type 4 (CR4), which are members 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 alpha (Hr1) was determined from cDNA clones. alpha (Hr1) contains an I domain, the inserted domain characteristic of a subset of mammalian alpha subunits, including the leukocyte integrin family, A phylogenetic tree constructed for the alpha subunits also supports the ancestral position of alpha (Hr1) in the monophyletic cluster of I domain-containing alpha integrins, The alpha (Hr1) gene shows hemocyte-specific expression on Northern blot analysis. Western blot analysis and immunocytochemical staining of the hemocytes of H. roretzi using anti-alpha (Hr1) Ab showed that alpha (Hr1) subunits exist on the surface of a subpopulation of phagocytic hemocytes, Furthermore, anti-alpha (Hr1) Ab inhibited C3-dependent phagocytosis, but not basic phagocytosis, 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 mediates phagocytosis in the primitive complement system of the ascidian.