, Ashland, OR). AZD3965 order In order to identify lysosomal proteases capable of initiating MHC II degradation, we screened a panel of cathepsins for their ability to proteolyse purified, detergent-solubilized human HLA-DR3, isolated from B-LCLs.

Initially, based on the notion that molecules with loosely bound peptides might be more susceptible to proteolysis, we used HLA-DR3 molecules isolated from the HLA-DM-deficient cell line 9.5.3. More than 70% of HLA-DR3 molecules isolated from the 9.5.3 cell line are loaded with CLIP.33 Degradation was monitored by SDS-PAGE and silver staining. Digestion of HLA-DR3 molecules with CatG at neutral pH generated two proteolytic intermediates, migrating at 15 and 18 kDa (Fig. 1a), which subsequent work showed to be derived from the DR β chain (see below).

The degradation of the β chain of HLA-DR3 was blocked (Fig. 1a) by addition of the CatG inhibitor,29 confirming that the observed Inhibitor Library β chain fragments were cleavage products generated specifically by CatG and not by contaminating proteases. No other cathepsin tested (D, L, S, H, and B) degraded HLA-DR3 at either neutral or endosomal pH (Fig. 1b and data not shown), although CatB and CatL degraded HLA-DM at pH 5·0 (see below) and CatD, H and S were active on myelin basic protein (MBP) and/or model substrates (data not shown). Thus, native HLA-DR3 molecules are susceptible to at most a small subset of lysosomal proteases,

including CatG, in vitro. HLA-DR molecules purified from DM-deficient cells, as well as insect cell-derived HLA-DR molecules, are mostly occupied by loosely bound peptides, and some fraction of these HLA-DR molecules may lack bound peptides. In order to test whether CatG susceptibility of HLA-DR was linked to occupancy of the peptide binding groove, we compared CatG cleavage of HLA-DR3 molecules purified from DM-null (5.2.4-DR3) and DM-expressing (8.1.6) B-LCLs. CatG treatment of 5.2.4-derived DR3 and 8.1.6-derived DR3 molecules resulted in similar fragmentation patterns, as visualized by Western blotting. Of the two fragments seen by silver staining, only the 18-kDa fragment is immunoreactive with the antiserum used (Fig. 2a). In addition, we tested whether the stable interaction between Silibinin HLA-DR1 and the influenza haemagglutinin (306–318) peptide34 influences CatG susceptibility. Soluble insect cell-derived DR1 (sDR1) was loaded to 80% saturation with AMCA-labelled influenza hemagglutinin-derived (AMCA-HA) peptide, free peptide was removed, and the resultant AMCA-HA/sDR1 complexes were digested with CatG in the presence or absence of a CatG inhibitor. The persistence of the AMCA-HA/sDR1 complex was then monitored by fluorescence resonance energy transfer (FRET), which occurs between tryptophan residues of sDR1 and the AMCA fluorophore attached to the HA peptide when the two are in close physical proximity.