CATHEPSIN-B CLEAVAGE AND RELEASE OF INVARIANT CHAIN FROM MHC CLASS-IIMOLECULES FOLLOW A STAGED PATTERN

A staged pattern of cathepsin B cleavage of MHC class II alpha,beta-bo und invariant (I-i) chain and release of fragments was defined. Charge -loss mutations in the I-i chain were created in three clusters of cat hepsin B putative cleavage sites R(78)K(80)K(83)K(86) (KK143)-K-137, a nd R(151)K(154). Products Of HLA-DR1 alpha,beta and wild type (WT) or mutant I-i genes, co-transfected into COS1 cells, were cleaved by cath epsin B and immunoprecipitated by antibodies either to MHC class II ch ains or to different I-i epitopes. In WT I-i, cathepsin B digestion ge nerated two forms of p21 I-i fragments: a p21 recognized by anti-C-ter minus antibodies and a p21 recognized by an antibody to a determinant near the N-terminus. C-terminal p21 was released from MHC class II alp ha,beta chains upon its formation while N-terminal p21 remained associ ated with MHC class II alpha,beta chains. Mutations at (KK143)-K-137 i nhibited the generation of N-terminal p21 by cathepsin B. Mutation at R(78)K(80)K(83)K(86) led to an accumulation of MHC class II-bound N-te rminal p21 without the appearance of MHC class II-bound p14, p10, and p6 fragments after cathepsin B digestion. These results indicate that cathepsin B cleaves wild type I-i first about (KK143)-K-137 to produce a MHC class II-associated N-terminal p21, which is then cleaved about R(78)K(80)K(83)K(86) to generate p14, p10 and finally p6 which still associates with MHC class II alpha,beta chains. This pattern of staged cleavage and release of I-i might be related to a concerted mechanism regulating the binding of antigenic peptides to MHC class II molecule s.