Comprehensive proteome analysis of lysosomes reveals the diverse function of macrophages in immune responses

Abstract

Phagocytosis and autophagy in macrophages have been shown to be essential to both innate and adaptive immunity. Lysosomes are the main catabolic subcellular organelles responsible for degradation and recycling of both extracellular and intracellular material, which are the final steps in phagocytosis and autophagy. However, the molecular mechanisms underlying lysosomal functions after infection remain obscure. In this study, we conducted a quantitative proteomics analysis of the changes in constitution and glycosylation of proteins in lysosomes derived from murine RAW 264.7 macrophage cells treated with different types of pathogens comprising examples of bacteria (Listeria monocytogenes, L. m), DNA viruses (herpes simplex virus type-1, HSV-1) and RNA viruses (vesicular stomatitis virus, VSV). In total, 3,704 lysosome-related proteins and 300 potential glycosylation sites on 193 proteins were identified. Comparative analysis showed that the aforementioned pathogens induced distinct alterations in the proteome of the lysosome, which is closely associated with the immune functions of macrophages, such as toll-like receptor activation, inflammation and antigen-presentation. The most significant changes in proteins and fluctuations in glycosylation were also determined. Furthermore, Western blot analysis showed that the changes in expression of these proteins were undetectable at the whole cell level. Thus, our study provides unique insights into the function of lysosomes in macrophage activation and immune responses.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; innate immunity; lysosome; macrophage; quantitative proteomics; tandem mass tag labeling.

Figure 1. Work flow of the sample preparation for mass spectrometry (MS) analysis

Mouse macrophage cell line RAW 264.7 were infected with L. m, HSV-1 and VSV or culture medium for 9 hours followed by harvest and purification. Lysosomes were purified by density gradient centrifugation and fractionation. Lysosome purity was detected with western blot and electron microscopy. High purity lysosomes were used in subsequent TMT based quantitative MS and analysis.

Figure 2. Subcellular distribution of the proteins in lysosomes

A., Distribution of the proteins identified in lysosome mass spectrometry according to their subcellular annotation. Proteins identified in lysosome were mapped to GO terms of cellular components using DAVID tool, number of proteins in each category was presented. The annotation of proteins localization in other subcellular regions was merged as the “Other” category. ER, endoplasmic reticulum; Mito, mitochondrion; Lyso/Endo, lysosome/endosome; Golgi, Golgi apparatus. B., Distribution of the proteins according to the significance of their enrichment in the subcellular localization. P-values were calculated by DAVID tool.

Figure 3. Proteomic alteration of lysosomes reflects the macrophage immune response

A., The change fold of lysosome specific proteins. The proteins abundances after infections were compared to the uninfected group. Lamp1 and Limp1 are lysosome marker proteins and serves as control. The dashed line equals to the average ratio of Lamp1 and Limp1, which indicates the average ratio of lysosomal structural proteins. The solid line equals to 1. B. The change fold of toll-like receptors. C. The change fold of inflammatory proteins. D. The change fold of MHC molecules.

Figure 4. Comparative and network analyses of upregulated proteins in lysosomes

A., Venn diagram showing the overlap of upregulated proteins (more than 2-fold) between each group. Numbers in each area represents the number of proteins in that category. The descriptions represent the subcellular localizations with highest enrichment in that category. B., Upregulated proteins were submitted to STRING for further analysis for protein-protein interaction. Networks were visualized by Cytoscape software. Proteins upregulated by different treatments are indicated by different colors. The proteins of related functions are labeled and indicated by colored backgrounds.

Figure 5. Analysis of lysosome-related glycoproteins

A., Subcellular distribution of glycoproteins in lysosomes. 193 Lysosome-related glycolproteins identified with mass spectrum were mapped to 179 GO terms of cellular components using DAVID tools, number of proteins in each category was presented. Further classification of intracellular localization is shown. ER, endoplasmic reticulum; Mito, mitochondrion; Lyso/Endo, lysosome/endosome; Golgi, Golgi apparatus, Ribo, ribosome. B., Dot plot showing the relative abundance of 300 glycosylated peptides and proteins that each peptide represented; the source of the peptides is indicated by colors. Red and blue dotted lines represent abundance ratios that equal to 2:1 and 1:2, respectively. The peptides that are more glycosylated (with a ratio 02C32:1) and less glycosylated (with a ratio 02C21:2) are labeled.

Figure 6. Further analysis of mass spectrometry (MS) results by Western blotting

A., Immunity-related proteins chosen for Western blot analysis. Relative abundance of 5 proteins quantified by mass spectrum were shown in table. Fold change > 2 is labeled in red (left panel). Right panel, similar amounts of protein extracts from control or infected cells were probed with antibodies against chosen proteins (Mib1, RhoB, Gbp5, Rsad2 and Oasl1). Lamp1 and GAPDH served as loading controls. B.-F., MS/MS data of represented peptides of chosen proteins. TMT-128 indicates control; TMT-129 indicates L. m infection; TMT-130 indicates HSV-1 infection; and TMT-131 indicates VSV infection.

Figure 7. Analysis of lysosome and autophagy activity by cell staining

Mouse macrophage cell line RAW 264.7 were infected with L. m, HSV-1 and VSV or culture medium for 9 hours. NBD-PZ/PI dual staining was performed for lysosome activity analysis, and Cyto-ID staining was performed for autophagy activity analysis. A.-B., Quantification of the activity of lysosomes A. or autophagy B. shows the differences between each group. C.-D., one of representative image of fluorescence histograms measured using a FACSCalibur showed comparable intensities of lysosome C. or autophagy D. signals in each group. Cells without staining were set as blank control and the gate was set referring to blank. Mean Values and standard deviations were calculated (*: p < 0.05, **: p < 0.01, ***: p < 0.001, Student's t-test).