Neutrophil ecto-Calreticulin: Implications for Immunity

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Calreticulin is best known as a multifunctional endoplasmic reticulum resident, calcium binding protein with chaperone function. However, calreticulin has been reported on the surface of some cells where it can participate in a variety cell functions. We have shown that calreticulin is present on the surface of circulating PMN and since calreticulin lacks a transmembrane domain, it must bind to an adaptor protein, that is resident in the PMN plasma membrane. Preliminary data indicate that the adaptor for calreticulin on PMN is resident in lipid rafts and GPI-anchored. Reciprocal immunoprecipitation-and immunoblotting with anti-calreticulin and anti-CD59 identify CD59 as the adaptor protein on PMN. Calreticulin is known to have immune functions and this proposal will focus on how PMN utilize surface calreticulin. PMN have a critical role in innate immunity as the primary phagocyte. We hypothesize that PMN use surface calreticulin to both prime the adaptive immune response by facilitating the processing of antigens; and limit the chance for an errant adaptive response by facilitating the PMN's clearance once it is apoptotic. The following objectives will provide information necessary for addressing the larger immunologic questions: 1) determine whether calreticulin uses its KDEL sequence for transport to the plasma membrane, and whether other factors such as stress and apoptosis affect transport and/or shedding; 2) define the reciprocal binding domains on calreticulin and CD59 using recombinant domains of calreticulin and sCD59 with selected mutations. Kinetic, real time binding will be monitored with surface plasmon resonance; 3) Define if ligating ecto-calreticulin induces unique intracellular signaling. The immune functions of PMN derived calreticulin will be largely determined by the biology of calreticulin-CD59 interaction. The results of the proposed calreticulin-CD59 studies will enable future studies on the potential dual roles of PMN ecto-calreticulin for priming adaptive immunity, a function that is relevant to vaccine design; and for augmenting the clearance of apoptotic PMN, a function that prevents autoimmunity.