2b), as detected by SDS-PAGE. Strikingly, there was only minimal loss of binding of the AMCA-HA peptide to HLA-DR1 upon digestion with CatG, and this slight loss was unaffected by the CatG inhibitor (Fig. 2c). Thus, peptide-loaded HLA-DR molecules are susceptible to CatG proteolysis, and cleavage of the β chain does not disrupt the integrity of the antigen-binding groove occupied by the peptide. To determine

the exact CatG cleavage site within the HLA-DR β chain, we performed N-terminal sequencing as well as peptide mapping Staurosporine supplier of the digestion products of purified soluble HLA-DR1 (sDR1). For these experiments we used sDR1 expressed in either insect cells or E. coli. Neither of these have a transmembrane domain and E. coli purified sDR1 is not glycosylated, which led to the fragments being smaller on gels (10 and 15 kDa). sDR1 expressed in insect cells (not shown) was used for identification of the N-terminal sequence of both fragments by Edman degradation (underlined italic sequence, Fig. 3a). The first residue of the larger fragment corresponds to the glycine (G) in position 1 of the mature protein. The first residue of the smaller fragment was selleck kinase inhibitor identified as glutamine (Q) at position 110. In order to define the boundaries

of both fragments, we also digested sDR1 expressed in E. coli (Fig. 3a), which is not glycosylated and was therefore used for MALDI-TOF analysis. The two bands were excised from a gel and digested with trypsin, Staphylococcus aureus V8 protease, or Arg-C protease. All peptides of these digests identified by mass spectrometry are indicated in black text in Fig. 3a. The peptide SFTVQRRVEPKVTVYPSKTQPL (underlined in Fig. 3a) was identified from a V8 digest and the peptide RVEPKVTVYPSKTQPL was identified from an Arg-C digest of the larger fragment, indicating that CatG did not not cleave after the arginine (R), but did cleave after leucine 109 (L109). Based on the masses of the two fragments and on the fact that their sequences were contiguous, these fragments appear to represent the complete β chain, which therefore has only a single CatG cleavage site. The cleavage site, between HLA-DRβ L109 and glutamine 110 (Q110,

L/Q), is located on a loop between fx1 and fx2 of the membrane-proximal, immunoglobulin-like domain, as indicated on the crystal structure of HLA-DR (Fig. 3b). To explore whether HLA-DR β chain polymorphism might influence CatG susceptibility, we first compared the amino acid sequences of several HLA-DR β chains [DRB1*0101 (DR1), DRB1*1501 (DR2b), DRB1*0301 (DR3), DRB1*0401, and DRB1*0404] and found conservation of the L/Q cleavage site (Fig. 4a). We then subjected various recombinant soluble HLA-DR allelic variants to digestion with CatG and used HLA-DR-specific rabbit serum (CHAMP) to measure residual levels of DRβ and detect the 18-kDa DRβ fragment (Fig. 4b). As predicted from sequence alignment, CatG degraded the β chain of all HLA-DR molecules tested.