26 The identity and purity of the isolated molecules were tested using sodium dodecyl sulphate–polyacrylamide selleck compound gel electrophoresis (SDS-PAGE) and Coomassie Blue or silver staining (not shown). CatG from human sputum or from neutrophils was purchased from Sigma-Aldrich (St Louis, MO); CatL and CatB were
purchased from Caltag (Burlingame, CA) or R & D Systems (Minneapolis, MN). Full-length or soluble MHC II or DM molecules (100 μg/ml) were incubated with different isolated cathepsins (50–100 ng protein) in reaction buffer [phosphate-buffered saline (PBS), pH 7·2, 2·5 mm dithiothreitol (DTT) or 0·1 m citrate, pH 5·0–6·0, and 2·5 mm DTT] at 37° for various times (routinely 2 hr). Digestion products were resolved by SDS-PAGE and analysed by silver staining. Soluble HLA-DR1 expressed in Schneider cells and purified26 was used for digestion with CatG. The digested products were separated Small molecule library price by SDS-PAGE followed by transfer to an Immulon-PSQ membrane (Millipore, Billerica, MA). The membrane was stained with Coomassie Blue and air-dried. The bands were cut out and submitted for N-terminal sequencing to the Protein and Nucleic Acid Facility (Stanford University School of Medicine). Soluble HLA-DR1 expressed in Escherichia coli (a kind gift
from L. Stern, Biochemistry and Molecular Pharmacology, University of Massachusetts, Worcester, MA) was used for digestion with CatG and stained with
Gelcode Blue (Pierce, Rockford, IL). Prominent CatG cleavage products were excised, reduced with DTT and alkylated with iodoacetamide. Duplicate gel pieces for each band were digested with either Arg-C or Glu-C (Sigma-Aldrich) and peptides were extracted using established protocols.30 Protease digests were subjected Methocarbamol to reverse-phase high-performance liquid chromatography (HPLC) separation and the HPLC eluant was spotted to MALDI target plates for MALDI-TOF/TOF mass spectrometry (MS) (Applied Biosystems 4700, Foster City, CA) analysis. Peptides were identified by tandem mass spectrometry (MS/MS) analysis utilizing the Mascot search engine. Recombinant soluble HLA-DR1 molecules were loaded with 100-fold excess of a 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-labelled variant of the influenza A hemagglutinin (HA) 307-319 peptide (AMCA-HA) (a kind gift from L. Stern) in PBS overnight at 37°. Free AMCA-HA was removed by centrifuging the binding reactions through spin columns (Sephadex G50 Superfine; BioRad, Hercules, CA) according to the manufacturer’s instructions. Binding stoichiometry was determined by absorption spectrophotometry at 280 and 350 nm, as described previously.31 HLA-DR molecules were 70–90% loaded with AMCA-HA. HLA-DR1/AMCA-HA complexes were incubated with 50 ng of CatG in CatG digestion buffer (PBS, pH 7·4, and 0·05% Tween-20).