Towards a human repertoire of monocytic lysosomal proteins.
The lysosomal compartment of human monocytic cells has never been investigated by a proteomic approach. By a combination of one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis, protein identification by N-terminal sequencing, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting and tandem mass spectrometry (MS/MS) peptide sequence analysis, we initiated an exhaustive study of the human lyososomal proteome, which aims at establishing a 2-D reference map of human soluble lyososomal proteins. Human monocytic U937 cells were induced to 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 mannose-6-phosphate receptor. Analysis of the purified fraction led to the preliminary 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 (leukocystatin and the human cellular repressor of E1A-stimulated genes) are described here for the first time as mannose-6-phosphate-containing proteins.
Journet
A
A
Laboratoire de Chimie des Protéines, CEA-Grenoble, France. ajournet@cea.fr
Chapel
A
A
Kieffer
S
S
Louwagie
M
M
Luche
S
S
Garin
J
J
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D008360:Mannosephosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism* / Q000648:ultrastructure; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020298:U937 Cells
D008360:Mannosephosphates; D011506:Proteins; C027693:mannose-6-phosphate