More efficient peptide binding to MHC class II molecules during cathepsin B digestion of I_i than after I_i release

The binding of a T cell-presented peptide to MHC class II α,β chains occurs as a concurrent process with the release of the associated invariant chain (I_i) by cathepsin B. I_i was digested by cathepsin B from solubilized, MHC class II α,β,I_i complexes in the presence of N-hydroxysuccinimidyl-4-azidobenzoate-conjugated, ¹²⁵I-labeled, influenza virus matrix (18-29) peptide. The peptide was crosslinked where it became bound. This HLA-DR1-restricted peptide bound about three times more efficiently to class II α,β chains of DR 1- positive B cells when present during cathepsin B digestion of I_i than when added afterward, also at pH 5.0. Binding was competed by similarly DR-restricted peptides. Cathepsin D cleaved I_i but did not enhance peptide binding. However, a trace level of cathepsin D, added to the assay for peptide binding in the presence of cathepsin B, further enhanced peptide binding about three times. These experiments support an hypothesis for the staged release of I_i fragments by cathepsin D and cathepsin B, catalyzing at one point the insertion of a peptide into the antigen binding site formed by class II α and β chains.