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.
Highlights
Lamellar bodies (LBs) are tubulovesicular secretory organelles of epithelial cells related to lysosomes
We analyzed a fraction enriched in epidermal LBs by LC-MS/MS, and we focused on cytoplasmic linker protein (CLIP)-170/restin, a protein described previously as mediating interactions between organelles and microtubules and being involved in vesicular trafficking [26]
In the LB-enriched fraction analyzed by immunoelectron microscopy, we were able to detect kallikrein-7 (Fig. 1d) and dermokine, two other proteins known to be secreted via LB [30, 31]
Summary
PROTEOMICS CHARACTERIZATION BY HIGH THROUGHPUT MASS SPECTROMETRY AND POSSIBLE INVOLVEMENT OF CLIP-170 IN THEIR TRAFFICKING/SECRETION*□S. 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. CLIP-170 Association with Lamellar Bodies of Human Epidermis lipase A2) that make the upper layers waterproof [8, 9], structural proteins like corneodesmosin that optimizes corneocyte cohesion [10], proteases (kallikreins and cathepsins), glycosidases and protease inhibitors (e.g. elafin, secretory leucocyte protease inhibitor, lympho-epithelial kazal-type-related inhibitor, and ␣2-macroglobulin-like) involved in the control of desquamation [11, 12], and antimicrobial peptides (defensins and cathelicidin) [13, 14], at the stratum granulosum/ stratum corneum interface. We showed that CLIP-170, together with one of its effectors, was localized on a subpopulation of LBs in normal human epidermis
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