Proteomic analysis of human lysosomes: Application to monocytic and breast cancer cells

Abstract

To date, about fifty lysosomal hydrolases have been identified, and most of them are targeted towards the lysosomes through a specific mannose-6-phosphate (M-6-P) tag. As more lysosomal hydrolases were expected to be discovered, we performed a proteomic study of soluble lysosomal proteins. Human cells were induced to secrete M-6-P proteins which were affinity purified on immobilized M-6-P receptor. The purified proteins were resolved by two-dimensional electrophoresis and analyzed by mass spectrometry. Twenty-two proteins were identified, among which 16 were well-known lysosomal hydrolases. The remaining species distributed as follows: epididymis-specific alpha-mannosidase is a new mannosidase homolog, cystatin F and CREG (cellular repressor of E1A-stimulated genes) were previously identified as M-6-P proteins (Journet et al., Electrophoresis 2000, 21, 3411-3419), and the last three, which are not hydrolases, were up to now considered as nonlysosomal. This two-dimensional reference map of human U937 M-6-P proteins was afterwards used for comparison with M-6-P proteins purified either from U937 differentiated into macrophage-like cells, or from human breast cancer MCF7 cells. Phorbol ester induced differentiation of U937 cells led to limited proteolytic cleavage or maturation of a discrete number of hydrolases. Five additional lysosomal hydrolases were identified from MCF7 samples. These results prove the usefulness of such a procedure to analyze the lysosomal content of various cell lines, to discover new M-6-P proteins, as well as to point towards unknown biological processes.

A Proteomic Analysis of Lysosomal Integral Membrane Proteins Reveals the Diverse Composition of the Organelle

“…A previous study identified 27 proteins from a soluble lysosomal fraction that bound to a mannose 6-phosphate affinity column (39). However, lysosomal membrane proteins are generally not transported to the organelle via this pathway as reflected by the highly sialylated state of their N-linked oligosaccharides.…”

HIV-1 Nef Triggers Macrophage Fusion in a p61Hck- and Protease-Dependent Manner

“…We wondered whether lysosome enzymes such as proteases might contribute to macrophage fusion, because they have been implicated in the formation of multinucleated myotubes (60,61). Therefore, we used a protease inhibitor mix, mostly non-cellpermeant, containing pepstatin A (inhibitor of aspartic proteases including cathepsin D), leupeptin (a broad inhibitor of cysteine proteases and cathepsin B), aprotinin (inhibitor of serine proteases), E64C (inhibitor of cysteine proteases such as cathepsins B, L, H, and K), and GM6001 (a broad metalloproteases inhibitor) (62)(63)(64). As shown in Fig.…”

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: The human mammary epithelial cell proteome

“…To tackle this problem, either subcellular fractionation or enrichment strategies of "examine less to see more" or "divide and conquer" have been more widely adopted for proteomic studies besides orthogonal fractionation prior to MS analysis. Functional protein complexes or organelles (e.g., ribosome [11], spliceosome [12], phagosome [13], lysosome [14], exosome [15], vesicle coat [16], microsome [17], mitochondria [18], Golgi apparatus [19], nucleolus [20], etc.) have been selectively isolated and comprehensively analyzed.…”