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αB-Crystallin Is Found in Detergent-resistant Membrane Microdomains and Is Secreted via Exosomes from Human Retinal Pigment Epithelial Cells*

Open AccessPublished:November 19, 2010DOI:https://doi.org/10.1074/jbc.M110.160135
      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.

      Introduction

      The small heat shock protein, αB-crystallin (αB)
      The abbreviations used are: αB, αB-crystallin; DRM, detergent-resistant membrane microdomain; MBCD, methyl β-cyclodextrin; RPE, retinal pigment epithelium; TEM, transmission electron microscopy.
      is a developmentally regulated gene product whose association with multiple pathologies of varied antecedents such as neurodegeneration, oncogenesis, and cataracts suggests a vital function for this protein (
      • Andley U.P.
      ,
      • Bhat S.P.
      ,
      • Horwitz J.
      ). Elevated levels of αB have been reported in Alexander, Alzheimer, and Parkinson diseases. It is expressed in astrocytes (
      • Renkawek K.
      • Voorter C.E.
      • Bosman G.J.
      • van Workum F.P.
      • de Jong W.W.
      ) and has been implicated in peripheral nerve myelination (
      • D'Antonio M.
      • Michalovich D.
      • Paterson M.
      • Droggiti A.
      • Woodhoo A.
      • Mirsky R.
      • Jessen K.R.
      ). It is known to be one of the main antigens involved in multiple sclerosis (
      • van Noort J.M.
      • Bajramovic J.J.
      • Plomp A.C.
      • van Stipdonk M.J.
      ). Its expression in a subset of basal-like breast carcinomas has led to its characterization as a novel oncoprotein (
      • Moyano J.V.
      • Evans J.R.
      • Chen F.
      • Lu M.
      • Werner M.E.
      • Yehiely F.
      • Diaz L.K.
      • Turbin D.
      • Karaca G.
      • Wiley E.
      • Nielsen T.O.
      • Perou C.M.
      • Cryns V.L.
      ). It is also a potential tissue biomarker for renal cell carcinoma (
      • Holcakova J.
      • Hernychova L.
      • Bouchal P.
      • Brozkova K.
      • Zaloudik J.
      • Valik D.
      • Nenutil R.
      • Vojtesek B.
      ). Interestingly, αB has also been shown to activate T cells (
      • Chou Y.K.
      • Burrows G.G.
      • LaTocha D.
      • Wang C.
      • Subramanian S.
      • Bourdette D.N.
      • Vandenbark A.A.
      ) and inhibit platelet aggregation (
      • Kozawa O.
      • Matsuno H.
      • Niwa M.
      • Hatakeyama D.
      • Kato K.
      • Uematsu T.
      ).
      In the eye, apart from its predominant presence in the ocular lens, αB was initially reported in primary cultures of human retinal pigment epithelium (RPE) (
      • Bhat S.P.
      • Nagineni C.N.
      ) and has since been shown to be expressed in the retina (
      • Andley U.P.
      ) and during early development of the rat eye in the embryonic RPE (
      • Nishikawa S.
      • Ishiguro S.
      • Kato K.
      • Tamai M.
      ). It is highly expressed in rod outer segments as well as in the rat RPE, following intense light exposures that lead to photoreceptor cell degeneration (
      • Sakaguchi H.
      • Miyagi M.
      • Darrow R.M.
      • Crabb J.S.
      • Hollyfield J.G.
      • Organisciak D.T.
      • Crabb J.W.
      ). In age-related macular degeneration, high concentrations of αB transcripts are found in microdissected retinal tissue juxtaposed with subretinal lipoprotein deposits, known as “drusen” (
      • Johnson P.T.
      • Brown M.N.
      • Pulliam B.C.
      • Anderson D.H.
      • Johnson L.V.
      ) and has been suggested to be a reliable marker of the progression of this neurodegeneration (
      • De S.
      • Rabin D.M.
      • Salero E.
      • Lederman P.L.
      • Temple S.
      • Stern J.H.
      ).
      At the molecular level, αB has been shown to have antiaggregation properties in vitro (
      • Horwitz J.
      ). When introduced into cells in culture, it protects them against apoptosis (
      • Andley U.P.
      ,
      • Kamradt M.C.
      • Chen F.
      • Cryns V.L.
      ) by interfering with caspase conversions (
      • Kamradt M.C.
      • Chen F.
      • Cryns V.L.
      ) and/or mitochondrial processes that are obligatory for cell death (
      • Mao Y.W.
      • Liu J.P.
      • Xiang H.
      • Li D.W.
      ,
      • Yaung J.
      • Jin M.
      • Barron E.
      • Spee C.
      • Wawrousek E.F.
      • Kannan R.
      • Hinton D.R.
      ). It has also been found in the nucleus (
      • Bhat S.P.
      • Hale I.L.
      • Matsumoto B.
      • Elghanayan D.
      ) and within the nucleus, has been detected in SC35 speckles (
      • van Rijk A.E.
      • Stege G.J.
      • Bennink E.J.
      • May A.
      • Bloemendal H.
      ). It interacts with 20 S proteasomal subunit C8/α7 (
      • Boelens W.C.
      • Croes Y.
      • de Jong W.W.
      ) and is reported to be involved in ubiquitin-dependent cyclin D1 proteolysis (
      • Barbash O.
      • Lin D.I.
      • Diehl J.A.
      ).
      Notwithstanding the abovementioned important activities reported for αB, a common thread that would explain the basic fundamental function of this protein remains to be established. For instance, this protein, in addition to being a component of extracellular age-related lipoprotein deposits in various neurodegenerations, is also known to activate T cells in multiple sclerosis (
      • Chou Y.K.
      • Burrows G.G.
      • LaTocha D.
      • Wang C.
      • Subramanian S.
      • Bourdette D.N.
      • Vandenbark A.A.
      ) and inhibit platelet aggregation (
      • Kozawa O.
      • Matsuno H.
      • Niwa M.
      • Hatakeyama D.
      • Kato K.
      • Uematsu T.
      ). The physiological basis of these seemingly extracellular activities (
      • Enomoto Y.
      • Adachi S.
      • Matsushima-Nishiwaki R.
      • Niwa M.
      • Tokuda H.
      • Akamatsu S.
      • Doi T.
      • Kato H.
      • Yoshimura S.
      • Ogura S.
      • Iwama T.
      • Kozawa O.
      ) of a protein known to be intracellular has not been addressed. We now show that it associates with detergent-resistant microdomains (DRMs) or lipid rafts and is secreted out of the cell via exosomes.

      DISCUSSION

      αB is known to be an intracellular protein; its primary sequence does not have any signatures, such as a signal peptide for secretion. It is, however, found associated with the Golgi in various tissues and cells (
      • Gangalum R.K.
      • Schibler M.J.
      • Bhat S.P.
      ,
      • Gangalum R.K.
      • Bhat S.P.
      ), including the ARPE19 cells (Fig. 1). In this investigation we demonstrate that αB is secreted out of the ARPE cells packaged in exosomes. It is significant that the two isoforms of αB, known to be related to phosphorylation (
      • Chiesa R.
      • McDermott M.J.
      • Spector A.
      ), are both seen inside the cell as well as outside in the culture medium (Fig. 3, arrows). This protein, however, does get O-GlcNAc on threonine 170 (
      • Roquemore E.P.
      • Chevrier M.R.
      • Cotter R.J.
      • Hart G.W.
      ), which explains its passage through Golgi. Importantly though, tunicamycin does not seem to impact the secretion of αB (supplemental Fig. S2D), thereby suggesting that this modification may not be important for its export out of the cell. Thus, it is conceivable that although O-GlcNAc-modified protein may have diverse destinations, possibly including the cell surface and the nucleus, the unmodified αB, a protein without a signal peptide, must come out of the cell, bypassing the endoplasmic reticulum-Golgi pathway via exosomes.
      It is interesting to examine the confocal microscope images presented in Fig. 1B. In addition to plasma membrane labeling with anti-αB, it seems that αB is localized to some larger structures in the cytoplasm that are not labeled by GM130. It is possible that these structures represent multivesicular bodies (
      • Mor-Vaknin N.
      • Punturieri A.
      • Sitwala K.
      • Faulkner N.
      • Legendre M.
      • Khodadoust M.S.
      • Kappes F.
      • Ruth J.H.
      • Koch A.
      • Glass D.
      • Petruzzelli L.
      • Adams B.S.
      • Markovitz D.M.
      ). In some of these structures, our preliminary data show colocalization of αB with CD63 (a marker of multivesicular bodies and exosomes). Importantly, this colocalization is susceptible to tunicamycin treatment,
      R. K. Gangalum and S. P. Bhat, unpublished data.
      suggesting that this association may be involved with part of the αB that is redistributed within the cell. However, the relevance of these findings to the export of αB via exosomes must await further investigations.
      Exosomes are known to be secreted from various cell types in vitro and have been found in various body fluids such as plasma, urine, amniotic fluid, bronchoalveolar lavage, synovial and cerebrospinal fluids (
      • Simpson R.J.
      • Jensen S.S.
      • Lim J.W.
      ). Exosomes have also been reported from ARPE19 cells (
      • McKechnie N.M.
      • Copland D.
      • Braun G.
      ,
      • Wang A.L.
      • Lukas T.J.
      • Yuan M.
      • Du N.
      • Tso M.O.
      • Neufeld A.H.
      ). It is interesting to note that the presence of αB has also been reported in human tear fluid (
      • May C.A.
      • Welge-Lüssen U.
      • Jünemann A.
      • Bloemendal H.
      • Lütjen-Drecoll E.
      ). It is possible that αB in the tear fluid is in exosomes.
      Although αB has been identified in the total proteome of RPE (
      • West K.A.
      • Yan L.
      • Shadrach K.
      • Sun J.
      • Hasan A.
      • Miyagi M.
      • Crabb J.S.
      • Hollyfield J.G.
      • Marmorstein A.D.
      • Crabb J.W.
      ), the reported “secretome” of RPE does not list αB as one of its proteins (
      • An E.
      • Lu X.
      • Flippin J.
      • Devaney J.M.
      • Halligan B.
      • Hoffman E.P.
      • Hoffman E.
      • Strunnikova N.
      • Csaky K.
      • Hathout Y.
      ). This may be because αB and other low abundance proteins may have escaped this analysis by virtue of being encased in membranous compartments as reported here (FIGURE 5, FIGURE 6, FIGURE 7, FIGURE 8, FIGURE 9). The resistance of αB to proteolysis (Fig. 8, C and D) establishes the presence of this protein in the lumen of the microvesicles. Additionally, the characteristic shape of the isolated exosomes, the known markers (FIGURE 5, FIGURE 7), and the predominant polarized secretion (Fig. 4) all authenticate the presence of αB in exosomes.
      Lipid rafts/DRMs are part of exosome biogenesis (
      • Rajendran L.
      • Simons K.
      ). They are cholesterol- and sphingolipid-rich membrane domains distinct from the rest of the more fluid plasma membrane. These structures are populated by a number of signaling protein molecules that give them their dynamic and possibly specific physiological functions (
      • Rajendran L.
      • Simons K.
      ,
      • Lingwood D.
      • Simons K.
      ). DRMs are assembled at the trans-Golgi domains and are then incorporated into various compartments, including the endosomes, the plasma membrane, and the exosomes (
      • Rajendran L.
      • Simons K.
      ). The data presented here, while explaining the relevance of the known association of αB with the Golgi membranes (
      • Gangalum R.K.
      • Schibler M.J.
      • Bhat S.P.
      ), also point to the mechanistic basis of its presence in the exosomes through its association with DRMs.
      The function of αB in microvesicles can only be speculated on at this time. Exosomes are potential extracellular signaling machines (
      • Pap E.
      • Pállinger E.
      • Pásztói M.
      • Falus A.
      ). For example αB-containing exosomes could stimulate T cells, known to be involved in the generation of the pathogenesis in multiple sclerosis (
      • Chou Y.K.
      • Burrows G.G.
      • LaTocha D.
      • Wang C.
      • Subramanian S.
      • Bourdette D.N.
      • Vandenbark A.A.
      ,
      • Bajramovic J.J.
      • Plomp A.C.
      • Goes A.
      • Koevoets C.
      • Newcombe J.
      • Cuzner M.L.
      • van Noort J.M.
      ). Based on the data presented in this investigation, we believe that αB-containing exosomes may represent a link in the generation of αB-specific T cells without the intervention of the release of αB from oligodendrocytes through apoptosis or injury.
      It is also known that αB imparts resistance to apoptosis in RPE (
      • Andley U.P.
      ); therefore, it is possible that exosomes loaded with this protein are taken up by nonexpressing cells (
      • Pap E.
      • Pállinger E.
      • Pásztói M.
      • Falus A.
      ), thus bypassing the need for de novo αB expression, an example of lateral transfer of molecular information. Notably, αB has also been shown to be present in interphotoreceptor matrix (
      • Hauck S.M.
      • Schoeffmann S.
      • Deeg C.A.
      • Gloeckner C.J.
      • Swiatek-de Lange M.
      • Ueffing M.
      ) that lies alongside the apical surface of the RPE in vivo.
      The relevance of αB secretion via apical face of ARPE to its accumulation in drusen on the basal side of RPE, in age-related macular degeneration, is not obvious. But we speculate that a pathological loss of polarity could overcome this physiological/physical separation. Alternatively, the drusen (
      • Wang A.L.
      • Lukas T.J.
      • Yuan M.
      • Du N.
      • Tso M.O.
      • Neufeld A.H.
      ,
      • Mullins R.F.
      • Russell S.R.
      • Anderson D.H.
      • Hageman G.S.
      ) may result from the secretory activity of αB-expressing cells, other than the RPE such as the microglia (
      • Raoul W.
      • Feumi C.
      • Keller N.
      • Lavalette S.
      • Houssier M.
      • Behar-Cohen F.
      • Combadière C.
      • Sennlaub F.
      ).
      Finally, the discovery of the association αB with DRMs (Fig. 6), the potential signal-organizing centers in the cell (
      • Lingwood D.
      • Simons K.
      ), may allow a deeper insight into functional import of the membrane association of α-crystallins, reported as early as 1979 (
      • Cobb B.A.
      • Petrash J.M.
      ,
      • Kibbelaar M.A.
      • Bloemendal H.
      ). This subcellular location in specialized lipid domains presents a vital avenue for future investigations that may yet reveal actual mechanistic details of the physiological role of this small heat shock protein, inside the cell and outside, in the exosomes.

      Acknowledgments

      We thank Garen Polatoglu and Josh Lee for technical help, Ankur Bhat for running the two-dimensional gel experiments, Jane Hu and Dean Bok for help with transepithelial resistance experiments and Grace Raposo for advice with isolation and identification of exosomes. We thank Drs. Joseph Horwitz and Dean Bok for reading the manuscript and for suggestions.

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