Characterization and crystallization of soluble human fc gamma receptor II(CD32) isoforms produced in insect cells

Fc gamma RII (CD32), the receptor for the Fc part of IgG, is responsible fo r the clearance of immunocomplexes by macrophages and plays a role in the r egulation of antibody production by B cells. To investigate the process of immunocomplex binding in terms of stoichiometry and stability of the Fc gam ma RII: IgG complex, we produced both Fc gamma RII isoforms (Fc gamma RIIa and Fc gamma RIIb) as soluble proteins in insect cells. The expressed prote ins could be purified in high yields and were biologically active as judged by their ability to bind IgG. Thus, the minor glycosylation performed by t he insect cells is not crucial for the binding of the usually highly glycos ylated Fc gamma RII to IgG. The dissociation constant of the sFc gamma RIIa : IgG-hFc complex was determined by fluorescence titration (K-D = 2.5 x 10( -7) M). Complementary sFc gamma RIIa antagonizes immunocomplex binding to B cells. Here sFc gamma RIIa showed a comparable dissociation constant (K-D = 1.7 x 10(-7) M) which was almost 10-fold lower than the constant for Fc g amma RIIb. The stoichiometry of the FcRIIa:IgC-hFc complex was determined b y equilibrium gel filtration and shows that IgG is able to bind alternative ly one or two Fc gamma RII molecules in a noncooperative manner. Furthermor e, in an ELISA-based assay the isotype specificity of various anti-Fc gamma RII monoclonal antibodies was measured as well as their ability to interfe re with the IgG recognition through its receptors. To further investigate t he molecular basis of the Fc gamma RII-ligand interaction, we crystallized Fc gamma RIIb. Trigonal crystals diffracted to 3 Angstrom and the structure solution is in progress.