Proteomic analysis of human lysosomes: application to monocytic and breast cancer cells.
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.
Laboratoire de Chimie des Protéines, INSERM ERIT-M 0201, Département Réponse et Dynamique Cellulaires, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France. ajournet@cea.fr
Agnès
A
Journet
Agnès
A
Chapel
Sylvie
S
Kieffer
Florence
F
Roux
Jérôme
J
Garin
D000818:Animals; D001943:Breast Neoplasms / Q000737:chemistry*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D008247:Lysosomes / Q000737:chemistry* / Q000201:enzymology; D008360:Mannosephosphates / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009000:Monocytes / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D020539:Sequence Analysis, Protein; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism
D008360:Mannosephosphates; D011506:Proteins; D020543:Proteome; C018589:lysosomal proteins; C027693:mannose-6-phosphate; D013755:Tetradecanoylphorbol Acetate