title: AlphaB-crystallin is found in detergent-resistant membrane microdomains and is secreted via exosomes from human retinal pigment epithelial cells.
abstract: αB-crystallin (αB) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of αB has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that αB is secreted from human adult retinal pigment epithelial cells via microvesicles (exosomes), independent of the endoplasmic reticulum-Golgi protein export pathway. The presence of αB in these lipoprotein structures was confirmed by its susceptibility to digestion by proteinase K only when exosomes were exposed to Triton X-100. Transmission electron microscopy was used to localize αB in immunogold-labeled intact and permeabilized microvesicles. The saucer-shaped exosomes, with a median diameter of 100-200 nm, were characterized by the presence of flotillin-1, α-enolase, and Hsp70, the same proteins that associate with detergent-resistant membrane microdomains (DRMs), which are known to be involved in their biogenesis. Notably, using polarized adult retinal pigment epithelial cells, we show that the secretion of αB is predominantly apical. Using OptiPrep gradients we demonstrate that αB resides in the DRM fraction. The secretion of αB is inhibited by the cholesterol-depleting drug, methyl β-cyclodextrin, suggesting that the physiological function of this protein and the regulation of its export through exosomes may reside in its association with DRMs/lipid rafts.
authors.affiliation: Jules Stein Eye Institute, UCLA, Los Angeles, California 90095, USA.
authors.forename: Rajendra K
authors.initials: RK
authors.lastname: Gangalum
authors.forename: Ivo C
authors.initials: IC
authors.lastname: Atanasov
authors.forename: Z Hong
authors.initials: ZH
authors.lastname: Zhou
authors.forename: Suraj P
authors.initials: SP
authors.lastname: Bhat
mesh_terms: D016764:Cell Polarity; D003902:Detergents / Q000494:pharmacology; D055354:Exosomes / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry*; D011506:Proteins / Q000032:analysis; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D038203:alpha-Crystallin B Chain / Q000032:analysis* / Q000378:metabolism; D047392:beta-Cyclodextrins
chemical_list: D003902:Detergents; D011506:Proteins; D038203:alpha-Crystallin B Chain; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin