Cathepsin B cleavage and release of invariant chain from MHC class II molecules follow A staged pattern

A staged pattern of cathepsin B cleavage of MHC class II α,β -bound invariant (I_i) chain and release of fragments was defined. Charge-loss mutations in the I_i, chain were created in three clusters of cathepsin B putative cleavage sites R⁷⁸K⁸⁰K⁸³K⁸⁶, K¹³⁷K¹⁴³, and R¹⁵¹K¹⁵⁴. Products of HLA-DR1 α,β and wild type (WT) or mutant I_i genes, co-transfected into COS1 cells, were cleaved by cathepsin B and immunoprecipitated by antibodies either to MHC class II chains or to different I_i epitopes. In WT I_i, cathepsin B digestion generated two forms of p21 I_i fragments: a p21 recognized by anti-C-terminus antibodies and a p21 recognized by an antibody to a determinant near the N-terminus. C-terminal p21 was released from MHC class II α,β chains upon its formation while N-terminal p21 remained associated with MHC class II α,β chains. Mutations at K¹³⁷K¹⁴³ inhibited the generation of N-terminal p21 by cathepsin B. Mutation at R⁷⁸K⁸⁰K⁸³K⁸⁶ led to an accumulation of MHC class II-bound N-terminal 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 K¹³⁷K¹⁴³ to produce a MHC class II-associated N-terminal p21, which is then cleaved about R⁷⁸K⁸⁰K⁸³K⁸⁶ to generate p14, p10 and finally p6 which still associates with MHC class II α,β 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 molecules.