Towards a human repertoire of monocytic lysosomal proteins

The lysosomal compartment of human monocytic cells has never been investiga ted by a proteomic approach. By a combination of one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis, protein identification by N-term inal sequencing, matrix assisted laser desorption/ionization-mass spectrome try (MALDI-MS) peptide mass fingerprinting and tandem mass spectrometry (MS /MS) peptide sequence analysis, we initiated an exhaustive study of the hum an lyososomal proteome, which aims at establishing a 2-D reference map of h uman soluble lyososomal proteins. Human monocytic U937 cells were induced t o secrete lysosomal soluble hydrolases by addition of NH4Cl in the culture medium. Since lysosomal soluble proteins are characterized by the presence of mannose-6-phosphate, they were purified on an affinity support bearing m annose-6-phosphate receptor. Analysis of the purified fraction led to the p reliminary identification of fifteen proteins, among which twelve are well- known lysosomal hydrolases, one is assumed to be lysosomal on the basis of sequence homology to cysteine proteinases of the papain family, and two (le ukocystatin and the human cellular repressor of E1A-stimulated genes) are d escribed here for the first time as mannose-6-phosphate-containing proteins .