Open Access Methodology article

A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells

Archana Vaithilingam12, Nicole Y Lai1, Ellen Duong1, Julie Boucau12, Yang Xu1, Mariko Shimada1, Malini Gandhi1 and Sylvie Le Gall12*

Author Affiliations

1 Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139, USA

2 Harvard Medical School, Boston, MA, USA

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BMC Cell Biology 2013, 14:35  doi:10.1186/1471-2121-14-35

Published: 9 August 2013

Additional files

Additional file 1: Figure S1:

Cysteine cathepsin activity is similar in live cells and lysate. Human primary CD4 T cells were grown in R10-IL2. 5 × 104 cells resuspended in PBS was added to each well. For lysate, the volume of the lysate derived from 5 × 104 cells was calculated and added to each well. When using inhibitors, E64 was added to the well and preincubated for 30 min at 37C. Following incubation, the activity of an omnicathepsin substrate was measured. Average and SD of 3 experiments.

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Additional file 2: Figure S2:

Cathepsin S and B substrates are cleaved by both enzymes. Cathepsin B and S activity was measured in crude PBMC lysate resuspended in pH 4.0 using fluoregenic subtrates. When using inhibitors, lysate was preincubated with Cathepsin S, D, K, B and an omnicathepsin inhibitor following which activity of the enzyme was measured. The average maximum slope of the curve derived from three replicates quantified in relative fluorescent units (RFU)/min is plotted.

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Additional file 3: Figure S3:

Degradation of peptides with crude lysate at pH 4.0 is equivalent to using purified endolysosomes for the oligopeptide B57-5ISW9-3. A) A long HIV peptide, 5-ISW9-3 (MVHQAISPRTLNAWVKV) was subjected to degradation using PBMC or purified endolysosomes at pH 4.0 or pH 5.5. The fragments produced were identified by mass spectrometry and the number of similar (light grey) and unique (dark grey) peptides between the two conditions was determined. Peptides that were not detected are indicated in grey letters. The surface of the peak of each peptide is indicated for each experimental condition. (B) The sum of all peptide peak intensities was determined and the distribution of peak intensities in similar (light grey) and unique (dark grey) fragments was calculated (upper). Of the total number of peptide fragments detected in each condition, the percentage of fragments with (dark grey) and without (light grey) the B57ISW9 epitope was calculated (middle panel). Similarly, the percentage of fragments that measured 13–19 amino acids in length (dark grey), 8–12 amino acids (white) and lesser than 7 amino acids (black) was determined. In all three cases, the average of the results from two MS runs of the same experiment was plotted.

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