Suraj P. Bhat

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

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.

Elevated Retina-Specific Expression of the Small Heat Shock Protein, αA-crystallin, Is Associated with Photoreceptor Protection in Experimental Uveitis

PURPOSE During the early phase of experimental autoimmune uveitis (EAU), before macrophages infiltrate the retina and uvea, photoreceptor mitochondrial oxidative stress, nitration of photoreceptor mitochondrial proteins, and release of cytochrome c have been observed. However, no apoptosis has been detected during this phase. In this study, alphaA-crystallin upregulation in the retina and its antiapoptotic protective role were evaluated in early EAU. METHODS Gene microarrays were first used to identify upregulated genes in retinas with early EAU. Among highly upregulated crystallins, alphaA was confirmed by real-time polymerase chain reaction and Western blot, and the site of upregulation was localized by immunohistochemistry. The association of alphaA-crystallin to nitrated cytochrome c and interaction with a procaspase-3 subunit was assayed. Photoreceptor apoptosis in alphaA knockout mice was compared with that in wild-type animals with EAU, by using the terminal transferase dUTP nick-end labeling assay and polymerase chain reaction. RESULTS In early EAU, alphaA-crystallin was increased 33-fold, and the site of increase was localized to the photoreceptor inner segments. This crystallin suppressed apoptosis by associating with the nitrated cytochrome c and p24. The association with nitrated cytochrome c, in particular, appeared to be restricted to nitrated cytochrome c, and thus, no association of non-nitrated cytochrome c was detected. The knockout mice showed signs of EAU development early and showed apoptosis in the retina; no such changes were seen in the wild-type control animals. CONCLUSIONS alphaA-Crystallin is highly upregulated in the retina during early EAU. This upregulation is localized primarily in the photoreceptor inner segments, the site of mitochondrial oxidative stress. Further, in early EAU, the photoreceptors preferentially use alphaA-crystallin to suppress mitochondrial oxidative stress-mediated apoptosis.

Crystallins, genes and cataract

Far from being a physical entity, assembled of inanimate structural proteins, the ocular lens epitomizes the biological ingenuity that sustains an essential and near-perfect physical system of immaculate optics. Crystallins (alpha, beta, and gamma) provide transparency by dint of their high concentration, but it is debatable whether proteins that provide transparency are any different, biologically or structurally, from those that are present in non-transparent structures or tissues. It is becoming increasingly clear that crystallins may have a plethora of metabolic and regulatory functions, both within the lens as well as outside of it. Alpha-crystallins are members of a small heat shock family of proteins and beta/gamma-crystallins belong to the family of epidermis-specific differentiation proteins. Crystallin gene expression has been studied from the perspective of the lens specificity of their promoters. Mutations in alpha-, beta-, and gamma-crystallins are linked with the phenotype of the loss of transparency. Understanding catalytic, non-structural properties of crystallins may be critical for understanding the malfunction in molecular cascades that lead to cataractogenesis and its eventual therapeutic amelioration.

Small Heat Shock Protein αB-Crystallin Is Part of Cell Cycle-dependent Golgi Reorganization

αB-Crystallin is a developmentally regulated small heat shock protein known for its binding to a variety of denatured polypeptides and suppression of protein aggregation in vitro. Elevated levels of αB-crystallin are known to be associated with a number of neurodegenerative pathologies such as Alzheimer disease and multiple sclerosis. Mutations in αB-crystallin gene have been linked to desminrelated cardiomyopathy and cataractogenesis. The physiological function of this protein, however, is unknown. Using discontinuous sucrose density gradient fractionation of post-nuclear supernatants, prepared from rat tissues and human glioblastoma cell line U373MG, we have identified discrete membrane-bound fractions of αB-crystallin, which co-sediment with the Golgi matrix protein, GM130. Confocal microscopy reveals co-localization of αB-crystallin with BODIPY TR ceramide and the Golgi matrix protein, GM130, in the perinuclear Golgi in human glioblastoma U373MG cells. Examination of synchronized cultures indicated that αB-crystallin follows disassembly of the Golgi at prometaphase and its reassembly at the completion of cytokinesis, suggesting that this small heat shock protein, with its chaperone-like activity, may have an important role in the Golgi reorganization during cell division.

αB‐Crystallin is expressed in kidney epithelial cell lines and not in fibroblasts

We have recently shown the presence of αB‐crystallin in non‐ocular tissues of diverse embryological origins such as the heart, brain, spinal cord, kidney, retina, etc. Using an αB‐crystallin‐specific antiserum and immunofluorescence, immunoblotting, immunoprecipitation and peptide mapping with Staphylococcus aureus protease, we demonstrate differential expression of αB‐crystallin in epithelial and fibroblast cell lines. αB‐Crystallin was detectable only in epithelial cell lines such as MDBK, MDCK, LLCPK1 and JTC‐12, and was not observed in two kidney fibroblast cell lines, one skin fibroblast cell line, and one corneal fibroblast cell line. Differential expression of the αB‐crystallin gene was also confirmed by Northern blot analysis of the RNAs isolated from these cell lines. These data suggest a cell‐type‐specific role for αB.

Ectopic expression of alpha B-crystallin in Chinese hamster ovary cells suggests a nuclear role for this protein.

alpha B-crystallin (alpha B) is known to be a cytosolic, small heat shock-like multimeric protein that has anti-aggregation, chaperone-like properties. The expression of the alpha B-crystallin gene is developmentally regulated and is induced by a variety of stress stimuli. Importantly, alpha B-crystallin expression is enhanced during oncogenic transformation of cells, in a number of tumors, and most notably, in many neurodegenerative disorders, including Alzheimers disease and multiple sclerosis. Other than its perceived role as a structural protein in the ocular lens, the actual function of alpha B-crystallin in cellular physiology remains unknown. We have stably transfected CHO cells with an inducible alpha B-cDNA-MMTV-promoter construct that allows the synthesis of recombinant alpha B-crystallin only upon exposure of these cells to dexamethasone. Using immunostaining and conventional and confocal microscopy, we have examined the subcellular distribution of the ectopically expressed alpha B-crystallin. We find that in addition to being in the cytoplasm, the protein resides in the nuclear interior in the interphase nucleus. Double labeling with anti alpha B-crystallin and anti-tubulin, concanavallin, and wheat germ agglutinin, respectively, revealed that during cell division alpha B-crystallin is excluded from condensed chromatin and the nascent nuclei. However, the protein again appears in the newly formed nuclei after the completion of cytokinesis suggesting a conditional, regulatory role for alpha B-crystallin in the nucleus.

Characterization and quantification of full-length and truncated Na, K-ATPase α1 and β1 RNA transcripts expressed in human retinal pigment epithelium

We have characterized cDNA clones encoding the alpha 1 and beta 1 subunits of Na,K-ATPase produced in the human retinal pigment epithelium (hRPE). In addition to isolating clones corresponding to known sequences of Na,K-ATPase subunits, we report hitherto unknown forms of Na,K-ATPase with unique deduced amino acid (aa) sequences in their C-termini. Truncated cDNA sequences were found for both the beta 1 and alpha 1 subunits. While the beta 1 sequence is truncated by two aa residues at the C terminus, in the alpha 1 sequence 342 aa have been replaced by a unique sequence containing only 44 aa. Interestingly, this new C-terminal polypeptide shows sequence similarities to the Ca(2+)-ATPase and contains consensus sequence elements for phosphorylation and cell adhesion, suggesting expression of Na,K-ATPase subunits with unique functions. Using reverse transcription-polymerase chain reaction, RNA sequences for alpha 1, beta 1 and their corresponding truncated isoforms were quantified. 4.0 x 10(5) alpha 1 and 2.3 x 10(5) beta 1 molecules were found per ng of mRNA from hRPE. Much lower levels were detected for truncated alpha 1 and beta 1 (3.6 x 10(3) and 2.7 x 10(3) molecules/ng, respectively). These data corroborate the expression of truncated transcripts coding for unique aa sequences in hRPE, and suggest that factors other than alpha 1 and beta 1 mRNA levels regulate the equimolar accumulation of alpha and beta subunits in the plasma membrane.

Pax-6 Expression in Posthatch Chick Retina during and Recovery from Form-Deprivation Myopia

The expression of Pax-6 in fully-differentiated chick retina remains largely confined to the amacrine and ganglion cell layers. In the developing posthatch chick retina, Pax-6 expression shows a biphasic pattern; a decrease by posthatch day 17 followed by a steady increase in the adult eye. Interestingly, we find that this biphasic expression of Pax-6 is reflected in the biphasic growth pattern of the posthatch chick eye, which is disrupted by form-deprivation myopia (FDM). We have now examined the pattern of Pax-6 accumulation in 3-day-old chick eyes subjected to 2 weeks of FDM followed by 2 weeks of recovery from FDM. Quantitative RT-PCR (with a homologous internal control) revealed that after 2 weeks of occlusion the contralateral non occluded eyes, the occluded eyes and the normal nonexperimental chick eyes did not show any drastic changes in the number of Pax-6 transcripts. The data obtained suggests that the contralateral eye does not represent a ‘normal’ control eye; similar but nonidentical changes are seen, at the molecular level, in both the contralateral and the occluded eyes. Comparisons with the control nonexperimental animals, however, are meaningful. Even after 2 weeks of recovery under normal light conditions, the occluded eyes do not seem to reach the same level of Pax-6 expression (number of molecules per mg tissue) as seen in normal control eyes, suggesting that exposure of the posthatch chick eye to FDM impedes developmental progression that normally culminates in emmetropia.

Expression and synthesis of the Na,K-ATPase β2 subunit in human retinal pigment epithelium

Na,K-ATPase in the retinal pigment epithelium (RPE) is apically localized, whereas in most other tissues this pump is found predominantly in the basolateral membrane domain. As part of our investigations into the molecular aspects of this pump in the RPE, we have cloned the cDNA and characterized the expression of the gene encoding the beta 2 subunit isoform of Na,K-ATPase in human, rat and bovine RPE and in the bovine choroid plexus. We have also detected the beta 2 isoform polypeptide in the human RPE (hRPE). Comparison of complete coding sequences derived from cloned cDNAs revealed that all beta 2 sequences from RPE, and the choroid plexus, differed uniformly at positions: P51/L, M121/I, and L148/R from the published sequences for human retina and liver. However, analysis of 10 RT-PCR clones derived from 5 fetal and 2 adult human retinas sequenced in our laboratory, revealed that only the P51/L residue was different with the hRPE beta 2 subunit sequence. Northern blot analysis indicated a 3.4-kb RNA transcript for the beta 2 subunit, a 4.5-kb RNA for the alpha 1 subunit and a doublet of 2.3 and 2.6 kb for the beta 1 subunit, respectively. alpha 1 (100 kDa), beta 1 (45 kDa) and beta 2 (65 kDa) isoforms were detected in hRPE extracts by immunoblotting. No alpha 2 and alpha 3 RNA transcripts were found in the hRPE. Quantification of beta 2 mRNA by RT-PCR revealed 2.7 x 10(5) molecules per ng of poly A+ RNA. This is similar to the beta 1 isoform levels reported previously from our laboratory. These data demonstrate the coexistence of significant amounts of alpha 1, beta 1 and beta 2 Na,K-ATPase subunits in the RPE. It is therefore reasonable to suggest that both alpha 1 beta 1 and alpha 1 beta 2 heterodimers are present in these cells.

Small Heat Shock Protein αA-Crystallin Prevents Photoreceptor Degeneration in Experimental Autoimmune Uveitis

The small heat shock protein, αA-crystallin null (αA−/−) mice are known to be more prone to retinal degeneration than the wild type mice in Experimental Autoimmune Uveoretinitis (EAU). In this report we demonstrate that intravenous administration of αA preserves retinal architecture and prevents photoreceptor damage in EAU. Interestingly, only αA and not αB-crystallin (αB), a closely related small heat shock protein works, pointing to molecular specificity in the observed retinal protection. The possible involvement of αA in retinal protection through immune modulation is corroborated by adaptive transfer experiments, (employing αA−/− and wild type mice with EAU as donors and Rag2−/− as the recipient mice), which indicate that αA protects against the autoimmune challenge by modulating the systemic B and T cell immunity. We show that αA administration causes marked reduction in Th1 cytokines (TNF-α, IL-12 and IFN-γ), both in the retina and in the spleen; notably, IL-17 was only reduced in the retina suggesting local intervention. Importantly, expression of Toll-like receptors and their associated adaptors is also inhibited suggesting that αA protection, against photoreceptor loss in EAU, is associated with systemic suppression of both the adaptive and innate immune responses.