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


RESULTS
In 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.


CONCLUSIONS
By 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.
Ragon Institute of MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139, USA.
Archana
A
Vaithilingam
Nicole Y
NY
Lai
Ellen
E
Duong
Julie
J
Boucau
Yang
Y
Xu
Mariko
M
Shimada
Malini
M
Gandhi
Sylvie
S
Le Gall
D000595:Amino Acid Sequence; D017951:Antigen Presentation / Q000502:physiology*; D000941:Antigens / Q000378:metabolism; D002403:Cathepsins / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism
D000941:Antigens; D011506:Proteins; C018589:lysosomal proteins; D002403:Cathepsins; D010447:Peptide Hydrolases