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

Abstract

BackgroundEndolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions.ResultsIn this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes.ConclusionBy using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides.

HIV Protease Inhibitor–Induced Cathepsin Modulation Alters Antigen Processing and Cross-Presentation

“…The pH of PBMCs cell extracts was reduced to 4-5.5 in order to specifically activate cathepsins (24). Similar PI-induced alterations of cathepsin activities were observed in PBMC extracts albeit with a lower fold change (unpublished data).…”

“…In order to analyze how PI-induced changes in cathepsin activities alter antigen processing in compartments involved in cross-presentation we used our in vitro antigen degradation assay (19, 24). We previously showed that the reduction of cellular extracts pH to 4 activates cathepsins, inhibits proteasome and aminopeptidase activities and mimics degradation conditions in purified endolysosomes (19, 24).…”

“…We previously showed that the reduction of cellular extracts pH to 4 activates cathepsins, inhibits proteasome and aminopeptidase activities and mimics degradation conditions in purified endolysosomes (19, 24). DC, macrophage and CD4 T cell whole cell extracts placed at acidic pH were used to degrade a synthetic 35-mer peptide in HIV-1 Gag p24 (MVHQAISPRTLNAWVKVVEEKAFSPEVIPMFAALS, aa10-44 in Gag p24) containing the HLA-B57-restricted epitopes ISW9 and KF11 (36).…”

“…2nmol of pure peptides were digested with 15μg of whole cell extracts at 37°C in pH4 degradation buffer (24). The degradation was stopped with 2.5μL of 100% formic acid and peptide fragments were purified by 5% trichloroacetic acid precipitation.…”

In vitro evaluation of digestive and endolysosomal enzymes to cleave CML‐modified Ara h 1 peptides

“…A recent article has demonstrated the utility of crude lysates from peripheral blood mononuclear cells (PBMCs) in characterizing endolysosomal protease activity during antigen processing (Vaithilingam et al. ).…”