title: Lamellar bodies of human epidermis: proteomics characterization by high throughput mass spectrometry and possible involvement of CLIP-170 in their trafficking/secretion.
abstract: Lamellar bodies (LBs) are tubulovesicular secretory organelles of epithelial cells related to lysosomes. In the epidermis, they play a crucial role in permeability barrier homeostasis, secreting their contents, lipids, a variety of hydrolases, protease inhibitors, and antimicrobial peptides, in the upper keratinocyte layers. The identification of proteins transported in epidermal LBs is still far from complete, and the way their secretion is controlled unknown. In this study, we describe the first proteomics characterization by nano-LC-MS/MS of a fraction enriched in epidermal LBs. We identified 984 proteins, including proteins known or thought to be secreted by LBs. Moreover 31 proteins corresponded to lysosomal components further suggesting that LBs are a new class of secretory lysosomes. Many of the newly found proteins could play a role in the epidermal barrier and desquamation (one acid ceramidase-like protein, apolipoproteins, glycosidases, protease inhibitors, and peptidases) and in LB trafficking (e.g. Rab, Arf, and motor complex proteins). We focus here on CLIP-170/restin, a protein that mediates interactions between organelles and microtubules. Western blotting confirmed the presence of CLIP-170 and its known effectors IQGAP1 and Cdc42 in the LB-enriched fraction. We showed, by confocal microscopy analysis of skin cryosections, that CLIP-170 was expressed in differentiated keratinocytes, first at the periphery of the nucleus then with a granular cytoplasmic labeling evocative of LBs. It was preferentially co-localized with Cdc42 and with the known LB protein cathepsin D. CLIP-170 was also largely co-localized with Rab7. This study strongly suggests a new function for CLIP-170, its involvement together with Cdc42 and/or Rab7 in the intracellular trafficking of LBs, and provides evidence that nano-LC-MS/MS combined with monodimensional electrophoresis separation constitutes a powerful method for identifying proteins in a complex mixture such as subcellular structures.
authors.affiliation: UMR5165 CNRS-University of Toulouse III, Institut Fédératif de Recherche Claude de Préval (INSERM-CNRS)-Université Paul Sabatier-Centre Hospitalier Universitaire de Toulouse) 31059 Toulouse, France.
authors.forename: Anne-Aurélie
authors.initials: AA
authors.lastname: Raymond
authors.forename: Anne
authors.initials: A
authors.lastname: Gonzalez de Peredo
authors.forename: Alexandre
authors.initials: A
authors.lastname: Stella
authors.forename: Akemi
authors.initials: A
authors.lastname: Ishida-Yamamoto
authors.forename: David
authors.initials: D
authors.lastname: Bouyssie
authors.forename: Guy
authors.initials: G
authors.lastname: Serre
authors.forename: Bernard
authors.initials: B
authors.lastname: Monsarrat
authors.forename: Michel
authors.initials: M
authors.lastname: Simon
mesh_terms: D001693:Biological Transport, Active; D002458:Cell Fractionation; D004817:Epidermis / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism / Q000648:ultrastructure; D053858:Metabolic Networks and Pathways; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000378:metabolism*; D015388:Organelles / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins / Q000378:metabolism
chemical_list: C101203:IQ motif containing GTPase activating protein 1; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D011506:Proteins; C018589:lysosomal proteins; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D020703:ras GTPase-Activating Proteins; C076744:cytoplasmic linker protein 170; D020764:cdc42 GTP-Binding Protein; D020691:rab GTP-Binding Proteins