Novel fragments of the Sjogren's syndrome autoantigens alpha-fodrin and type 3 muscarinic acetylcholine receptor generated during cytotoxic lymphocyte granule-induced cell death

Objective. To determine whether the Sjogren's syndrome autoantigens alpha - fodrin and the type 3 muscarinic acetylcholine receptor (M3R) are cleaved d uring cytotoxic lymphocyte granule-induced death, to yield novel fragments. Methods. Primary salivary gland epithelial cells, human salivary gland cell s, and HeLa cells were incubated with granule contents. The susceptibility to cleavage and the generation of novel fragments of Sjogren's syndrome aut oantigens in this form of apoptosis was assessed by immunoblotting. Cleavag e of M3R was further characterized by assays performed on the M3R molecule generated by in vitro translation. Results. This study demonstrated that alpha -fodrin was uniquely cleaved du ring cytotoxic lymphocyte granule-induced cell death, generating a 155-kd f ragment distinct from those generated by caspase 3 in other forms of apopto sis. The study also demonstrates that M3R (which is restricted in expressio n to the peripheral autonomic organs) was efficiently cleaved by granzyme B (but not by caspases) at several sites, both in vitro and in intact cells. This is the first description of cleavage of a transmembrane autoantigen b y granzyme B. Conclusion. The observation that both ubiquitously expressed autoantigens ( e.g., alpha -fodrin, La, and nuclear mitotic apparatus protein) and tissue- restricted autoantigens (e.g., M3R) targeted in Sjogren's syndrome are spec ifically cleaved by granzyme B, generating unique fragments, strongly sugge sts that a common biochemical event (novel autoantigen cleavage during gran ule-induced epithelial cell death) is responsible for selecting this appare ntly unconnected group of molecules for a high-titer autoantibody response. The data focus attention on the role of cytotoxic lymphocytes in the initi ation and propagation of Sjogren's syndrome.