A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells
© Vaithilingam et al.; licensee BioMed Central Ltd. 2013
Received: 15 April 2013
Accepted: 6 August 2013
Published: 9 August 2013
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.
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.
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.
KeywordsEndolysosome Antigen processing Proteases Cathepsins Protein degradation Primary cells Mass spectrometry T cell epitope production MHC HIV
Endolysosomes are cellular organelles that play a key role in protein turnover and homeostasis of the cell. They are made up of a complex set of enzymes that break down proteins, lipids and sugars that are all essential for normal functioning of the cell . A genetic mutation in some of these endolysosomal proteins can lead to disorders such as Gaucher’s disease, Niemann pick’s disease, and Fabry disease [2–5]. Endolysosomes also play a key role in antigen presentation by processing large proteins into epitopes that can be presented by MHC Class I and Class II molecules [6–8]. Therefore it is important to develop methodologies suitable to study specific endolysosomal functions in relevant cells and tissues.
Several methods have been used to study their functions in the cell. The approaches taken to study endolysosome contribution to protein degradation include using recombinant cathepsins in peptide degradation assays , studying the effect of cathepsin knock-out mice on the epitope repertoire , and using purified endolysosomes from human tissue as a source of proteases for protein degradation assay . In the case of endolysosomal storage disorders, knock-in mice expressing a mutated form of an enzyme have been used to study the role of specific proteins in disease .
Each of the above mentioned approaches have some caveats. Endolysosomes comprise a complex set of proteases that are still not completely characterized. Studying the role of a single protease and their contribution to antigen presentation using recombinant proteins is not representative of the complex milieu inside the organelle. Gene knock-out or siRNA studies are helpful in identifying the precise role of a particular protein but do not help in addressing the interactions between the different proteins in the organelle. Though using purified endolysosomes will address the above-mentioned concerns, it involves a laborious, multistep process and results in a much lesser yield. In studies involving limited amount of material such as primary cells from patients, it may not be feasible to purify endolysosomes.
In this study, we describe a simple approach to test the contribution of endolysosomal proteases to protein degradation. Since endolysosomal proteases are active in acidic pH , we tested this characteristic by varying the pH in crude peripheral blood mononuclear cell (PBMC) lysate by varying the pH and showed that endolysosomal cathepsin activity can be specifically activated at acidic pH. This simple yet efficient approach enabled us to test the contribution of endolysosomal proteases to several cellular functions using primary cells and therefore is physiologically relevant. We compared these low pH extracts to purified endolysosomes and found that both methodologies produced similar protease activities and peptide degradation kinetics. Here, we exemplify the role of endolysosomes in antigen processing and highlight the potential usefulness of this method to study the function of endolysosomal enzymes in other functions and human diseases.
Differential centrifugation of PBMC lysate yields pure endolysosomes
Our previous studies established that short HIV peptides are variably sensitive to cytosolic degradation, a property that contributes to the efficient presentation of MHC-I epitopes . We reasoned that due to differences in the peptidase content between cytosol (proteasome, aminopeptidases, endopeptidases) and endolysosomes (cathepsins) we may use differential peptide degradation as an additional approach to assess cytosolic and endolysosomal fraction purity, as well as a readout of endolysosomal hydrolytic activities that does not involve fluorogenic substrates. The percentage of acid phosphatase activity in each fraction was used to determine the number of cells that yielded endolysosomes in each fraction. For example, if fraction 4 consisted of 30% of the total acid phosphatase activity, it was assumed that 30% of the initial number of PBMC yielded the endolysosomal proteins in that fraction. Using this information to calculate equivalent amounts of cytosol and endolysosome fractions purified from the same PBMC, we compared the degradation of two HIV p24 Gag-derived epitopes: B27KK10 (KRWIILGLNK, 131-140aa, a ten amino acid long HIV peptide restricted by HLA-B27), and B57KF11 (KAFSPEVIPMF, 30-40aa, an 11-amino acid long HIV peptide restricted by the HLA-B57)  (Figure 1D). The disappearance of the peptide was monitored by RP-HPLC, where the amount of peptide is proportional to the surface area under its peak. Upon degradation the peak area reduced and additional peaks corresponding to degradation products appeared . The two HIV peptides were degraded faster in endolysosomes compared to the cytosol. The half-life of B57KF11 was 36 min in the cytosol and decreased to 8 min in endolysosomes. Similarly, the half-life of B27KK10 was greater than 60 min in the cytosol and reduced to 38 min in the endolysosomes, suggesting that the distinct sets of peptidases present in cytosol and endolysosomes differently degraded the two peptides, also providing further evidence of the purity of the PBMC fractions.
Cathepsin activities can be measured using crude lysate at acidic pH
PBMC cathepsins involved in protein processing degrade peptides at acidic pH
Endolysosomes and crude lysate at acidic pH degrade peptides with the same kinetics and produce similar fragments
Endolysosomes contain proteins that are involved in diverse functions in the cell . Loss of activity in several of these proteins leads to disease [2–5]. Current approaches to study endolysosomal proteins are not sufficient to comprehensively understand the role of the organelle in cellular functions and using purified organelles is not feasible in studies involving access to limited amount of material such as primary cells and tissues. Because several endolysosomal proteases are activated in acidic pH , we have utilized this characteristic to develop a simple approach to study the role of these proteases in peptide degradation using crude PBMC lysate.
Most studies involving endolysosomal protease activity use recombinant purified cathepsins , manipulations such as gene knockouts in in vitro cell cultures  or purified endolysosomes from cells or tissues . In some cases, the results obtained are not the most physiologically relevant and require further validation in primary human tissues. Using the methodology described here, it is possible to assess the activity of one or simultaneously, many endolysosomal enzymes using crude lysate prepared from primary cells. Also, by modulating the pH, specific enzymes can be activated and cross-reactivity from other cytosolic proteins can be avoided while allowing to follow protein degradation in endolysosomal compartments. By using appropriate substrates for each enzyme a novel diagnostic for lysosomal protein disorders may be envisioned.
Another area of research that will benefit greatly from this assay is antigen presentation. Antigen presentation takes place in two different pathways inside the cell; the endogenous cytosolic pathway predominantly involved in class I presentation and the exogenous endolysosomal pathway involved in class II and class I epitope cross-presentation . One of the questions that has yet to be addressed in antigen presentation is the characteristics that govern efficient presentation of epitopes from a pathogen-derived protein endocytosed in cells. To date, the precise nature by which long pathogen-derived proteins are degraded in endocytic compartments to produce epitope length fragments is poorly defined. Despite the critical role of protein degradation in endolysosomes to elicit immune responses the nature of peptides cross-presented by MHC-I and MHC-II is not well defined in part due to the lack of adequate assays. Class II epitopes are not as well characterized as class I epitopes because of the limited knowledge of anchor residues within the MHC class II protein and looser binding of peptides onto MHC-II. Using our assay, it will be possible to study the degradation of pathogen-derived proteins or vaccine immunogens in crude extracts at neutral vs. acidic pH and identify epitopes produced in cytosol and endolysosomal compartments by mass spectrometry. Using these peptide fragments in in vivo antigen presentation assays and assaying for CD4 or CD8 T cell stimulation will further help to define class I and class II epitopes that are produced after intracellular processing and cross-presentation.
Vaccine immunogens (inactivated pathogens, purified proteins, peptides, viral vectors) mostly enter dendritic cells through endolysosomes for degradation followed by presentation of peptides by MHC-I and MHC-II leading to the priming of CD8 T cell responses and CD4 T helper cell responses . Knowledge of epitopes that are well defined and preserved within cellular compartments will enable efficient vaccine design. This is especially important in light of chimeric vaccine immunogens (which includes vaccines made of protein fragments separated by linkers, peptides derived from conserved regions of proteins that do not undergo mutations or peptides that include both wild type and mutated versions separated by linkers), so as to include peptides that will be efficiently processed and presented on the cell surface . With increased interest in targeted vaccines, wherein, antigens can be engineered to target specific compartments, once again, knowledge of stable epitopes that are produced efficiently in each of the targeted compartments is of immense importance. The methodology described here will facilitate these studies and bypass the need of intensive labor, access to large amounts of primary human cells and complicated techniques.
In summary, we have shown that using crude PBMC cellular extracts at low pH, endolysosomal protease activity can be measured in a simple and efficient manner, results of which can be used in several applications. We demonstrated that by varying the pH in crude cellular extracts, we could specifically activate certain cathepsins at each pH and thereby monitor protease activity over the course of endolysosomal maturation. We demonstrated the usefulness of this assay in the context of antigen processing and showed comparable results to using purified endolysosomes.
Buffy coats from blood donated by anonymous healthy donors were purchased from Massachusetts General Hospital. Partners Human Research Committee (Boston, MA) approved the use of buffy coats under protocol no. 2005P001218.
Reagents and antibodies
Antibodies for Lysosome Associated Membrane Protein 1 (LAMP1) (sc-20011) and cathepsin S (sc-271619) were purchased from Santa Cruz, beta actin (ab8227) and calnexin (ab22595) from Abcam and proteasome α 7 subunit (BML-PW8110-0100) from Enzo Lifesciences. All chemicals were purchased from Sigma and fluorogenic substrates were purchased from Enzo lifesciences. Highly purified HIV peptides were purchased from Massachusetts General Hospital peptide core facility or from Biosynthesis.
Endolysosomes were purified from crude PBMC lysate by differential centrifugation. PBMCs were isolated from human buffy coats using Ficoll centrifugation . Cells resuspended in extraction buffer at a concentration of 5 × 108 cells/ml  were lysed using glass beads as in a previous study done in our laboratory . The cell lysate was centrifuged at 1000g for 10 min at 4°C. The supernatant was transferred to a new tube and centrifuged at 20,000 g for 20 min at 4°C. The resulting pellet consists of endoplasmic reticulum, mitochondria and endolysosomes. The supernatant corresponds to purified cytosolic fraction. A step gradient ranging from 23% to 8% was built using an Optiprep based endolysosome isolation kit (Sigma, LYSISO1). The tubes were centrifuged at 100,000 g in a Beckmann Coulter tabletop centrifuge at 4°C. 120 μl fractions were collected and further analyzed for endolysosomal enrichment.
Following endolysosomal purification, 10μl of each fraction and the post nuclear supernatant were loaded on a 4-12% SDS PAGE gradient gel (Invitrogen) run at a constant 185V. The proteins were transferred to a PVDF membrane (Fisher Scientific) using a semi-dry blotting system (Bio-Rad) for 35 min at 20V. The membrane was blocked using a blocking buffer  for 1 hour at RT followed by incubation with the primary antibodies overnight at 4C. The membrane was washed three times, 10 min each with 0.1% TBS Tween followed by incubation with secondary antibodies, Goat anti rabbit (Licor, 926–68071) and Donkey anti mouse (Licor, 926–32212) for 1 hour at RT. The membrane was washed again for three times in wash buffer and imaged using a Licor imaging system.
Enzyme activity assay
Activities of proteases were tested using a fluorogenic enzyme substrate specific for each endolysosomal cathepsin . For endolysosomal purification samples, 3 μl of each fraction was added to 96 well plates. For analyzing cathepsin activity in different pH, 3 μg of crude PBMC lysate was added to each well. Appropriate amount of the enzyme specific substrate (omni-cathepsin 50 μM, chymotryptic activity of proteasome 100 μM and aminopeptidase 12.5 μM, cathepsin S 50 μM, cathepsin L 50 μM, cathepsin D 10 μM, cathepsin K 50 μM, cathepsin B 50 μM) resuspended in 200 μl of buffer (20 mM HEPES, 50 mM potassium acetate, 5 mM magnesium chloride, 1 mM ATP, 1mM DTT for proteasome, same buffer without ATP and DTT for aminopeptidase and 50 mM sodium chloride, 50 mM potassium phosphate, 2 mM EDTA and 2 mM DTT for cathepsins) were added to each well containing extracts or buffer (to control for background fluorescence). Fluorescence was measured every 5 minutes for 3 hours using a Victor-3 plate fluorescence reader (Perkin Elmer, Boston, MA) as described before . The resulting fluorescence curves were plotted over time. For each condition, the maximum rate of fluorescence emission was calculated after background correction, which is equivalent to the maximal rate of proteolytic hydrolysis. To verify the specificity of each substrate hydrolysis, crude lysate was pre-incubated with appropriate amounts of peptidase inhibitors: ZFL-COCHO 10 μM to inhibit Cathepsin S, pepstatin 100μM to inhibit Cathepsin D, BML-244 10 μM to inhibit Cathepsin K, E64 50 μM to inhibit all cysteine cathepsins, MG 132 10 μM to inhibit proteasome and bestatin 12.5 μM to inhibit aminopeptidase for 30 minutes at room temperature before addition of the fluorogenic substrate. Maximum slopes of the fluorescence over time curves in the presence or absence of inhibitors showed a 35-100% specific inhibition of each activity (data not shown).
Acid phosphatase assay
The presence of acid phosphatase in endolysosomal fractions was confirmed using a kit from Sigma (CS0740). 10 μl of each fraction was incubated with the substrate in the provided substrate buffer for 10 min at 37°C. 200 μl of the stop solution (2M sodium hydroxide) was added and the absorbance at 405 nM was measured. A blank without any lysate serving as a negative control and pure enzyme serving as a positive control was also included in the experiment. The amount of acid phosphatase present was calculated using the formula, Units/ml = (A405(sample)-A405(blank)) × 0.05 × 0.3 × DF/A405(standard)x time x Venz, where DF is dilution factor and Venz is volume of the pure enzyme.
Peptide degradation assay
The assay is adapted from previously published studies from our laboratory . 8 nM of peptide was incubated with 30 μg of crude PBMC lysate in 250 μl of degradation buffer (20 mM HEPES, 1 mM magnesium chloride, 137 mM potassium acetate, 1 mM ATP) that was adjusted to the appropriate pH. In studies involving purified endolysosome, an acid phosphatase assay was performed on the fractions and the amount of acid phosphatase activity was used to calculate the total number of cells from which each fraction was derived. Using this approach, equivalent amount of cytosol and endolysosome that were derived from the same number of cells were used in peptide degradation assays. For the mass spectrometry analysis of peptide fragments, the level of omni-cathepsin enzyme activity was assessed using the activity assay and volume of crude lysate and purified endolysosomes containing similar levels of the enzyme activity was used in the degradation assay. When using inhibitors, the compound was added to the crude lysate before addition of the peptide and incubated at room temperature for 30 minutes. The mixture was incubated at 37°C and at each time point 50 μl were collected and the reaction was stopped by adding 2.5 μl of 100% trifluoroacetic acid. Another 50 μl of the degradation buffer was added to each sample and the amount of peptide remaining was analyzed by RP-HPLC.
Mass spectrometry analysis of peptide fragments
Following peptide degradation, the peptide fragments produced were enriched using 10% trichloroacetic acid precipitation and identified by in-house mass spectrometry analysis as previously described . After diluting the product to 500 femtomole in 80% water, 15% acetonitrile, 5% trifluoroethanol, the peptides were resolved in RP-HPLC on a C18 column (Nano-LC Eksigent) and electrosprayed on an Orbitrap discovery mass spectrometer (Thermo). The peptides were identified using Proteome Discoverer software (Thermo) and the surface intensity of each peptide peak corresponded to the amount of peptide present. The peptide fragments were aligned using the Jalview software and number of fragments present in both the conditions were used to calculate the percentage of similar peptides detected. The experiments were repeated twice using two different endolysosomal preparations and degradation products of each experiment were analyzed twice with the mass spectrometer.
Peripheral blood mononuclear cells
Major histocompatibility complex
Lysosomal associated membrane protein 1
Human immunodeficiency virus
Reversed phase high performance liquid chromatography.
This project was funded by the National Institute of Allergy and Infectious diseases (NIAID) (RO1 A1084753) and a Ragon Innovation award to SLG.
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