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.

FIGURE 1.

αB is a Golgi membrane-associated protein in ARPE cells. A, sucrose density gradient fractionation of the postnuclear homogenate of confluent ARPE cells is shown. The top panel is an immunoblot showing the predominant presence of αB in Golgi membrane fractions (black line), as indicated by GM130 distribution (second panel). The bottom three panels show immunoblots with calnexin, cytochrome c (Cyto C), and Na⁺/K⁺-ATPase used as endoplasmic reticulum, mitochondria, and plasma membrane markers, respectively. Protein markers are shown on the left in the top panel. Ext, total cell extracts before fractionation. B, confocal images show perinuclear colocalization of αB (red) and the Golgi membrane-specific protein GM130 (green). This colocalization is lost upon treatment of cells with brefeldin A (BFA) (lower panel). Scale bar, 20 μm. Approximately 40% of αB colocalizes with GM130; in brefeldin A-treated cells this falls to 2%.

FIGURE 2.

Time course of the appearance of αB in culture medium. The cells were grown to 70% confluence in serum containing medium, washed three times, and incubated with fresh prewarmed serum-free medium (SF) or with fresh prewarmed serum-containing (+serum) medium (SM) for the indicated time periods. Separate flasks (three for each time point) were used. One flask per time point was used for cell counting. αB was detected by immunoblotting (insets) of the concentrated medium and quantified using recombinant αB standards on the same blots. For serum-free medium, protein, 40 μg/lane, was analyzed for each time point. For serum-containing medium, however, comparable volumes were used, which contained more than 200 μg of total protein per lane (because of the presence of serum). The percentage of total αB in the medium has been plotted and shown in supplemental Fig. S1D.

FIGURE 3.

Secreted αB is similar to intracellular protein. αB in the total cell extract and in the medium was characterized by two-dimensional gel electrophoresis and immunoblotting. The arrows indicate two differentially phosphorylated isoforms of the protein. *, respective aliquots run only on the second-dimension gel. Arrow on the right indicates the direction of second-dimension SDS-PAGE. Protein markers are shown on the left.

FIGURE 4.

αB is released predominantly from the apical face of human ARPE cells. Human ARPE19 cells were grown on Transwell culture inserts (see “Experimental Procedures”) for about 3 weeks with regular monitoring of the trans-epithelial resistance. A, in transepithelial resistance (TER) measurements of ARPE cells, the data are represented as mean ± S.E. (error bars, n = 3). The resistance across the ARPE monolayer is measured using EVOM chop stick electrode and calculated (TER = Ω × cm²). B, the experiment was started by changing the medium on the apical as well as basal compartments and waiting 12 h before the medium was collected from respective compartments, concentrated, and immunoblotted. Note preferential presence of αB in the apical medium. Data from two different experiments are shown.

FIGURE 5.

αB is secreted in exosomes. A, sucrose density gradient fractionation profile of the concentrated medium. Various proteins including αB were detected by immunoblotting of the gradient fractions. The top panel shows that there are two distinct pools of αB in the gradient, the predominant membrane-associated fraction in the middle and the minor fraction on the top. In this panel the protein markers are on the left. The presence of flotillin-1 and α-enolase in second and third panels, respectively suggests the existence of exosomes in these fractions. Caveolin-1 and LAMP-1 were not detected in the gradient. The vertical panels (on the right) show positive controls with total cell extracts (Ext). B, acetylcholinesterase (AChE) activity in various fractions of the gradient shown in A. That αB is vesicle-associated is corroborated by the presence of acetylcholinesterase activity in these fractions.

FIGURE 6.

αB is associated with lipid rafts/DRMs. A, total cell extracts were fractionated on OptiPrep gradients (29). Top panel (−MBCD) shows that a significant amount of αB is associated with DRMs or lipid rafts (indicated by a line, fractions 2–4). In the lower panel cells were exposed to MBCD (5 mm, +MBCD) for 6 h prior to fractionation. Note that with +MBCD, both αB and caveolin-1 are significantly lost from the DRM fractions. There are differential effects of this drug on Hsp70 and flotillin-1. B, fractionation of the medium from the MBCD-treated and control cells on OptiPrep gradients. The concentrated medium without any further manipulations was loaded into the bottom of the gradient and fractionated as in A. There is no detectable αB in the medium analyzed from +MBCD cells. However, flotillin-1 and Hsp70 (both reduced) are still seen in the gradient, which has now shifted toward the bottom of the gradient, suggesting disruption of vesicle formation. The right column shows positive controls for all the antibodies used. Ext, total cell extract; Med, medium before fractionation.

FIGURE 7.

Exosomes isolated from ARPE cells contain αB, Hsp70, flotillin-1 and α-enolase. A, complete immunoblot with anti-αB. Protein standards are on the left (top panel). The bottom three panels show only the relevant part of the respective immunoblots. E, total cell extract; P, exosome pellet obtained from the medium; S, supernatant from the exosome pellet. Lane E contains 40 μg of protein; lanes P and S contain comparable volumes of resuspended exosome pellet and concentrated supernatant (medium), respectively. B, TEM of uranyl acetate-stained exosome pellet. Note the characteristic saucer-shaped vesicles. C, size distribution of exosomes seen in B.

FIGURE 8.

TEM-immunogold labeling of the native exosomes and resistance of exosomal αB to proteinase K digestion. A and B, exosome pellets processed for immunogold labeling without permeabilization. Double labeling was done using antibodies, anti-αB and anti-Hsp70. Hsp70 was used as a marker because this protein has been shown to be present in exosomes. Goat anti-rabbit IgG conjugated to 12-nm (arrows) and 18-nm (arrowheads) gold particles was used for the detection of αB and Hsp70, respectively. Not all vesicles are labeled by both antibodies. Some vesicles are unlabeled, which is expected because the vesicular structures are intact. In the case of αB, the antibody is against the C terminus (25), which may be more accessible (3). C, susceptibility of αB to proteinase K in concentrated culture medium before isolation of exosomes. Digestion was followed by immunoblotting of the reactions. The protein in the medium is resistant to proteinase K digestion in the absence of Triton X-100 (lane 2). D, immunoblot of isolated exosome pellet subjected to proteinase K digestion in the absence (lanes 1 and 2) and in the presence (lanes 3 and 4) of Triton X-100 (0.25%, lane 3; 0.5%, lane 4).

FIGURE 9.

TEM-immunogold labeling of exosomes exposed to Triton X-100. A, collapsed vesicle (permeabilized with 0.02% Triton-X100) immunogold-labeled with anti-αB. There is enhanced labeling of αB (12-nm gold particles, arrows) because of increased accessibility of the protein. B, TEM of Triton X-100-treated exosome pellet labeled with anti-Hsp70 (18-nm gold, arrows). See supplemental Fig. S4 for preimmune controls.