Proteomic Analysis of S-Acylated Proteins in Human B Cells Reveals Palmitoylation of the Immune Regulators CD20 and CD23

S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation

The potentials of MS-based subproteomic approaches in medical science: The case of lysosomes and breast cancer

International audienc

Biochemical characterization and lysosomal localization of the mannose-6-phosphate protein p76 (hypothetical protein LOC196463)

Most soluble lysosomal proteins carry Man6P (mannose 6-phosphate), a specific carbohydrate marker that enables their binding to cellular MPRs (Man6P receptors) and their subsequent targeting towards the lysosome. This characteristic was exploited to identify novel soluble lysosomal proteins by proteomic analysis of Man6P proteins purified from a human cell line. Among the proteins identified during the course of the latter study [Journet, Chapel, Kieffer, Roux and Garin (2002) Proteomics, 2, 1026–1040], some had not been previously described as lysosomal proteins. We focused on a protein detected at 76 kDa by SDS/PAGE. We named this protein ‘p76’ and it appeared later in the NCBI protein database as the ‘hypothetical protein LOC196463’. In the present paper, we describe the identification of p76 by MS and we analyse several of its biochemical characteristics. The presence of Man6P sugars was confirmed by an MPR overlay experiment, which showed the direct and Man6P-dependent interaction between p76 and the MPR. The presence of six N-glycosylation sites was validated by progressive peptide-N-glycosidase F deglycosylation. Experiments using N- and C-termini directed anti-p76 antibodies provided insights into p76 maturation. Most importantly, we were able to demonstrate the lysosomal localization of this protein, which was initially suggested by its Man6P tags, by both immunofluorescence and sub-cellular fractionation of mouse liver homogenates

Étude de p76, une nouvelle protéine mannose-6-phosphate : caractérisations biochimiques, localisation lysosomale et approche de la fonction

La protéine p76 ( hypothetical protein LOC196463 ) a été identifiée au laboratoire lors d'une analyse protéomique ciblant les protéines mannose-6-phosphate de lignées cellulaires humaines U937 et MCF7. Ce rapport de thèse présente l'étude de cette protéine concernant certaines de ses caractéristiques biochimiques, sa localisation intracellulaire et l'approche de sa fonction. Ainsi, nous avons mis en évidence la présence de 6 N-glycosylations, ainsi que la présence effective de sucres mannose-6-phosphate. Une maturation protéolytique des précurseurs de la forme humaine et murine de p76 a été observée ; les chaînes issues de ces clivages ont été en partie caractérisées à l'aide des anticorps développés au laboratoire. L'étude de sa localisation intracellulaire par immunofluorescence et par fractionnements subcellulaires réalisés sur du foie de souris indique clairement que p76 est localisée dans les lysosomes. Enfin, p76 ayant une homologie de séquence avec une phospholipase B nouvellement caractérisée de Dictyostelium discoideum, des tests fonctionnels ont été mis en œuvre pour détecter une telle activité pour la protéine recombinante hp76-myc, mais sans succès. En revanche, une expérience de fat blot a montré que hp76-myc se lie à la cardiolipine, un phospholipide particulier des membranes mitochondriales et bactériennesThe protein p76 ( hypothetical protein LOC196463 ) was identified some years ago in our laboratory during the course of a proteomic analysis of mannose-6-phosphate proteins purified from human cell lines. The present thesis describes the biochemical characterisation and the intracellular localisation of p76, as well as some functional tests carried out with recombinant p76. We demonstrated that human p76 sequence bears 6 N-glycosylations and that mannose-6-phosphate sugars were present. Proteolytic maturation of human and murine p76 precursors was observed; a few intermediate forms were partially characterised thanks to anti-p76 antibodies which were developed in the laboratory. Most importantly, we were able to demonstrate the lysosomal localisation of this protein by both immunofluorescence and sub-cellular fractionation of mouse liver homogenates. As p76 shows a significant sequence homology to a recently cloned phospholipase B from Dictyostelium discoideum, some functional tests were performed, but no phospholipase activity could be detected with recombinant hp76-myc. However, a fat blot experiment showed that hp76-myc binds cardiolipin, a particular phospholipid enriched in mitochondrial and bacterial membranesGRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Affinity Purification of Soluble Lysosomal Proteins for Mass Spectrometric Identification

International audienc

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

International audienc

Towards a human repertoire of monocytic lysosomal proteins

International audienceThe 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 NH$_4$Cl 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

Major involvement of cathepsin B in the intracellular proteolytic processing of exogenous IgGs in U937 cells

International audienc

Nucleotide sequence of the gene for the immunity protein to colicin A. Analysis of codon usage of immunity proteins as compared to colicins

International audienc

Calcium influx mediates the chemoattractant-induced translocation of the arrestin-related protein AdcC in Dictyostelium

International audienc