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

// Yanpan Gao 1,* , Yanyu Chen 1,* , Shaohua Zhan 1 , Wenhao Zhang 2 , Feng Xiong 1 and Wei Ge 1 1 Department of Immunology, National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Dongcheng, Beijing, China 2 MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, China * These authors have contributed equally to this work Correspondence to: Feng Xiong, email: // Wei Ge, email: // Keywords : innate immunity, lysosome, macrophage, quantitative proteomics, tandem mass tag labeling, Immunology and Microbiology Section, Immune response, Immunity Received : July 14, 2016 Accepted : December 31, 2016 Published : January 08, 2017 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.