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Raquel L. Lieberman - One of the best experts on this subject based on the ideXlab platform.
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molecular architecture and modifications of full length Myocilin
Experimental Eye Research, 2021Co-Authors: Mackenzie D Martin, Dustin J E Huard, Ricardo C Guerreroferreira, Ishani M Desai, Brett M Barlow, Raquel L. LiebermanAbstract:Abstract Myocilin, a modular multidomain protein, is expressed broadly in the human body but is best known for its presence in the trabecular meshwork extracellular matrix, and Myocilin misfolding is associated with glaucoma. Despite progress in comprehending the structure and misfolding of the Myocilin olfactomedin domain, the structure and function of full-length Myocilin, and contextual changes in glaucoma, remain unknown. Here we expressed and purified milligram-scale quantities of full-length Myocilin from suspension mammalian cell culture (Expi293F), enabling molecular characterization in detail not previously accessible. We systematically characterized disulfide-dependent and -independent oligomerization as well as confirmed glycosylation and susceptibility to proteolysis. We identified oligomeric states with glycosylation sites that are inaccessible to enzymatic removal. Low-resolution single particle 2D class averaging from conventional transmission electron microscopy imaging confirms an extended arrangement of tetramers, truncated products consistent with dimers, and a higher-ordered state consistent with octamer. Taken together, our study reveals new Myocilin misfolded states and layers of intrinsic heterogeneity, expands our knowledge of olfactomedin-family proteins and lays the foundation for a better molecular understanding of Myocilin structure and its still enigmatic biological function.
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recombinant antibodies recognize conformation dependent epitopes of the leucine zipper of misfolding prone Myocilin
Journal of Biological Chemistry, 2021Co-Authors: Athena C Pattersonorazem, Shannon E. Hill, Ahlam Qerqez, Laura R Azouz, Minh Thu, Lisa A Schildmeyer, Jennifer A Maynard, Raquel L. LiebermanAbstract:Recombinant antibodies with well-characterized epitopes and known conformational specificities are critical reagents to support robust interpretation and reproducibility of immunoassays across biomedical research. For Myocilin, a protein prone to misfolding that is associated with glaucoma and an emerging player in other human diseases, currently available antibodies are unable to differentiate among the numerous disease-associated protein states. This fundamentally constrains efforts to understand the connection between Myocilin structure, function, and disease. To address this concern, we used protein engineering methods to develop new recombinant antibodies that detect the N-terminal leucine zipper structural domain of Myocilin and that are cross-reactive for human and mouse Myocilin. After harvesting spleens from immunized mice and in vitro library panning, we identified two antibodies, 2A4 and 1G12. 2A4 specifically recognizes a folded epitope while 1G12 recognizes a range of conformations. We matured antibody 2A4 for improved biophysical properties, resulting in variant 2H2. In a human IgG1 format, 2A4, 1G12, and 2H2 immunoprecipitate full-length folded Myocilin present in the spent media of human trabecular meshwork (TM) cells, and 2H2 can visualize Myocilin in fixed human TM cells using fluorescence microscopy. These new antibodies should find broad application in glaucoma and other research across multiple species platforms.
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molecular insights into Myocilin and its glaucoma causing misfolded olfactomedin domain variants
Accounts of Chemical Research, 2021Co-Authors: Raquel L. Lieberman, Minh ThuAbstract:ConspectusNumerous human disorders arise due to the inability of a particular protein to adopt its correct three-dimensional structure in the context of the cell, leading to aggregation. A new addition to the list of such protein conformational disorders is the inherited subtype of glaucoma. Different and rare coding mutations in Myocilin, found in families throughout the world, are causal for early onset ocular hypertension, a key glaucoma risk factor. Myocilin is expressed at high levels in the trabecular meshwork (TM) extracellular matrix. The TM is the anatomical region of the eye that regulates intraocular pressure, and its dysfunction is associated with most forms of glaucoma. Disease variants, distributed across the 30 kDa olfactomedin domain (mOLF), cause Myocilin to be sequestered intracellularly instead of being secreted to the TM extracellular matrix. The working hypothesis is that the intracellular aggregates cause a toxic gain of function: TM cell death is thought to lead to TM matrix dysfunction, hastening elevated intraocular pressure and subsequent vision loss.Our lab has provided molecular underpinnings for Myocilin structure and misfolding, placing Myocilin-associated glaucoma within the context of amyloid diseases like Alzheimer and diabetes. We have dissected complexities of the modular wild-type (WT) Myocilin structure and associated misfolded states. Our data support the model that full-length WT Myocilin adopts a Y-shaped dimer-of-dimers conferred by two different coiled-coil regions, generating new hypotheses regarding its mysterious function. The mOLF β-propellers are paired at each tip of the Y. Disease-associated variants aggregate because mOLFs are less stable, leading to facile aggregation under physiological conditions (37 °C, pH 7.2). Mutant Myocilin aggregates exhibit numerous characteristics of amyloid in vitro and in cells, and aggregation proceeds from a partially folded state accessed preferentially by disease variants at physiological conditions. Interestingly, destabilization is not a universal consequence of mutation. We identified counterintuitive, stabilizing point variants that adopt a non-native structure and do not aggregate; however, these variants have not been identified in glaucoma patients. An ongoing effort is predicting the consequence of any given mutation. This effort is relevant to interpreting data from large-scale sequencing projects where clinical and family history data are not available. Finally, our work suggests avenues to develop disease-modifying precision medicines for Myocilin-associated glaucoma.
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How does a protein's structure spell the difference between health and disease? Our journey to understand glaucoma-associated Myocilin.
'Public Library of Science (PLoS)', 2019Co-Authors: Raquel L. LiebermanAbstract:Over 20 years ago, alterations to the protein Myocilin were confirmed to be linked to a heritable form of the prevalent eye disease, glaucoma, and 10 years ago, my lab set out to develop a deeper understanding of Myocilin in its normal and diseased state. We have made strides in understanding how genetic mutations in Myocilin likely lead to disease, but unlocking Myocilin's biological function is still an elusive goalpost. Is normal Myocilin unimportant in the human body? Are scientists using the wrong methods to study Myocilin biology? Here, I discuss my scientific journey into understanding one small part of the fascinating organ that is the eye
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epitope mapping of commercial antibodies that detect Myocilin
Experimental Eye Research, 2018Co-Authors: Athena C Pattersonorazem, Shannon E. Hill, Michael P. Fautsch, Raquel L. LiebermanAbstract:Abstract The presence of Myocilin is often used in the process of validating trabecular meshwork (TM) cells and eye tissues, but the antibody reagents used for detection are poorly characterized. Indeed, for over a century, researchers have been using antibodies to track proteins of interest in a variety of biological contexts, but many antibodies remain ill-defined at the molecular level and in their target epitope. Such issues have prompted efforts from major funding agencies to validate reagents and combat reproducibility issues across biomedical sciences. Here we characterize the epitopes recognized by four commercial Myocilin antibodies, aided by structurally and biochemically characterized Myocilin fragments. All four antibodies recognize enriched Myocilin secreted from human TM cell media. The detection of Myocilin fragments by ELISA and Western blot reveal a variety of epitopes across the Myocilin polypeptide chain. A more precise understanding of Myocilin antibody targets, including conformational specificity, should aid the community in standardizing protocols across laboratories and in turn, lead to a better understanding of eye physiology and disease.
Stanislav I. Tomarev - One of the best experts on this subject based on the ideXlab platform.
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Myocilin regulates metalloprotease 2 activity through interaction with timp3
Investigative Ophthalmology & Visual Science, 2017Co-Authors: Myung Kuk Joe, Raquel L. Lieberman, Naoki Nakaya, Stanislav I. TomarevAbstract:Purpose To elucidate functions of wild-type Myocilin, a secreted glycoprotein associated with glaucoma. Methods Lysates of mouse eyes were used for immunoprecipitation with affinity-purified antibodies against mouse Myocilin. Shotgun proteomic analysis was used for the identification of proteins interacting with Myocilin. Colocalization of Myocilin and tissue inhibitor of metalloproteinases 3 (TIMP3) in different eye structures was investigated by a multiplex fluorescent in situ hybridization and immunofluorescent labeling with subsequent confocal microscopy. Matrix metalloproteinase 2 (MMP2) activity assay was used to test effects of Myocilin on TIMP3 inhibitory action. Results TIMP3 was identified by a shotgun proteomic analysis as a protein that was coimmunoprecipitated with Myocilin from eye lysates of wild-type and transgenic mice expressing elevated levels of mouse Myocilin but not from lysates of transgenic mice expressing mutated mouse Myocilin. Interaction of Myocilin and TIMP3 was confirmed by coimmunoprecipitation of Myocilin and TIMP3 from HEK293 cells transiently transfected with cDNAs encoding these proteins. The olfactomedin domain of Myocilin is essential for interaction with TIMP3. In the eye, the main sites of Myocilin and TIMP3 colocalization are the trabecular meshwork, sclera, and choroid. Using purified proteins, it has been shown that Myocilin markedly enhanced the inhibitory activity of TIMP3 toward MMP2. Conclusions Myocilin may serve as a modulator of TIMP3 activity via interactions with the Myocilin olfactomedin domain. Our data imply that in the case of Myocilin null or some glaucoma-causing mutations, inhibitory activity of TIMP3 toward MMP2 might be reduced, mimicking deleterious mutations in the TIMP3 gene.
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Myocilin is involved in ngr1 lingo 1 mediated oligodendrocyte differentiation and myelination of the optic nerve
The Journal of Neuroscience, 2014Co-Authors: Heung Sun Kwon, Naoki Nakaya, Mones Abuasab, Hong Sug Kim, Stanislav I. TomarevAbstract:Myocilin is a secreted glycoprotein that belongs to a family of olfactomedin domain-containing proteins. Although Myocilin is detected in several ocular and nonocular tissues, the only reported human pathology related to mutations in the Myocilin gene is primary open-angle glaucoma. Functions of Myocilin are poorly understood. Here we demonstrate that Myocilin is a mediator of oligodendrocyte differentiation and is involved in the myelination of the optic nerve in mice. Myocilin is expressed and secreted by optic nerve astrocytes. Differentiation of optic nerve oligodendrocytes is delayed in Myocilin-null mice. Optic nerves of Myocilin-null mice contain reduced levels of several myelin-associated proteins including myelin basic protein, myelin proteolipid protein, and 2′3′-cyclic nucleotide 3′-phosphodiesterase compared with those of wild-type littermates. This leads to reduced myelin sheath thickness of optic nerve axons in Myocilin-null mice compared with wild-type littermates, and this difference is more pronounced at early postnatal stages compared with adult mice. Myocilin also affects differentiation of oligodendrocyte precursors in vitro. Its addition to primary cultures of differentiating oligodendrocyte precursors increases levels of tested markers of oligodendrocyte differentiation and stimulates elongation of oligodendrocyte processes. Myocilin stimulation of oligodendrocyte differentiation occurs through the NgR1/Lingo-1 receptor complex. Myocilin physically interacts with Lingo-1 and may be considered as a Lingo-1 ligand. Myocilin-induced elongation of oligodendrocyte processes may be mediated by activation of FYN and suppression of RhoA GTPase.
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Myocilin regulates cell proliferation and survival
Journal of Biological Chemistry, 2014Co-Authors: Myung Kuk Joe, Heung Sun Kwon, Radu Cojocaru, Stanislav I. TomarevAbstract:Myocilin, a causative gene for open angle glaucoma, encodes a secreted glycoprotein with poorly understood functions. To gain insight into its functions, we produced a stably transfected HEK293 cell line expressing Myocilin under an inducible promoter and compared gene expression profiles between Myocilin-expressing and vector control cell lines by a microarray analysis. A significant fraction of differentially expressed genes in Myocilin-expressing cells was associated with cell growth and cell death, suggesting that Myocilin may have a role in the regulation of cell growth and survival. Increased proliferation of Myocilin-expressing cells was demonstrated by the WST-1 proliferation assay, direct cell counting, and immunostaining with antibodies against Ki-67, a cellular proliferation marker. Myocilin-containing conditioned medium also increased proliferation of unmodified HEK293 cells. Myocilin-expressing cells were more resistant to serum starvation-induced apoptosis than control cells. TUNEL-positive apoptotic cells were dramatically decreased, and two apoptotic marker proteins, cleaved caspase 7 and cleaved poly(ADP-ribose) polymerase, were significantly reduced in Myocilin-expressing cells as compared with control cells under apoptotic conditions. In addition, Myocilin-deficient mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis as compared with wild-type mesenchymal stem cells. Phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in Myocilin-expressing cells compared with control cells. Elevated phosphorylation of ERK1/2 was also observed in the trabecular meshwork of transgenic mice expressing 6-fold higher levels of Myocilin when compared with their wild-type littermates. These results suggest that Myocilin promotes cell proliferation and resistance to apoptosis via the ERK1/2 MAPK signaling pathway.
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Myocilin mediates myelination in the peripheral nervous system through erbb2 3 signaling
Journal of Biological Chemistry, 2013Co-Authors: Heung Sun Kwon, Myung Kuk Joe, Mones Abuasab, Thomas V Johnson, Jun Zhang, Chichao Chan, Stanislav I. TomarevAbstract:The glaucoma-associated gene, Myocilin, is expressed in ocular and non-ocular tissues including the peripheral nervous system, but its functions in these tissues remain poorly understood. We demonstrate that in sciatic nerve, Myocilin is expressed in Schwann cells with high concentrations at the nodes of Ranvier. There, Myocilin interacts with gliomedin, neurofascin, and NrCAM, which are essential for node formation and function. Treatment of isolated dorsal root ganglion cultures with Myocilin stimulates clustering of the nodal proteins neurofascin and sodium channel Nav1.2. Sciatic nerves of Myocilin null mice express reduced levels of several myelin-associated and basal membrane proteins compared with those of wild-type littermates. They also demonstrate reduced myelin sheath thickness and partial disorganization of the nodes. Myocilin signaling through ErbB2/3 receptors may contribute to these observed effects. Myocilin binds to ErbB2/ErbB3, activates these receptors, and affects the downstream PI3K-AKT signaling pathway. These data implicate a role for Myocilin in the development and/or maintenance of myelination and nodes of Ranvier in sciatic nerve.
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Myocilin stimulates osteogenic differentiation of mesenchymal stem cells through mitogen activated protein kinase signaling
Journal of Biological Chemistry, 2013Co-Authors: Heung Sun Kwon, Thomas V Johnson, Stanislav I. TomarevAbstract:Myocilin is a secreted glycoprotein that is expressed in ocular and non-ocular tissues. Mutations in the Myocilin gene may lead to juvenile- and adult-onset primary open-angle glaucoma. Here we report that Myocilin is expressed in bone marrow-derived mesenchymal stem cells (MSCs) and plays a role in their differentiation into osteoblasts in vitro and in osteogenesis in vivo. Expression of Myocilin was detected in MSCs derived from mouse, rat, and human bone marrow, with human MSCs exhibiting the highest level of Myocilin expression. Expression of Myocilin rose during the course of human MSC differentiation into osteoblasts but not into adipocytes, and treatment with exogenous Myocilin further enhanced osteogenesis. MSCs derived from Myoc-null mice had a reduced ability to differentiate into the osteoblastic lineage, which was partially rescued by exogenous extracellular Myocilin treatment. Myocilin also stimulated osteogenic differentiation of wild-type MSCs, which was associated with activation of the p38, Erk1/2, and JNK MAP kinase signaling pathways as well as up-regulated expression of the osteogenic transcription factors Runx2 and Dlx5. Finally, cortical bone thickness and trabecular volume, as well as the expression level of osteopontin, a known factor of bone remodeling and osteoblast differentiation, were reduced dramatically in the femurs of Myoc-null mice compared with wild-type mice. These data suggest that Myocilin should be considered as a target for improving the bone regenerative potential of MSCs and may identify a new role for Myocilin in bone formation and/or maintenance in vivo.
Ernst R. Tamm - One of the best experts on this subject based on the ideXlab platform.
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increased expression of olfactomedin 1 and Myocilin in podocytes during puromycin aminonucleoside nephrosis
Nephrology Dialysis Transplantation, 2011Co-Authors: Daniela C Bohr, Rudolf Fuchshofer, Marcus Koch, Michaela Kritzenberger, Ernst R. TammAbstract:Background. The olfactomedin domain proteins Olfm-1 and Myocilin are expressed in podocytes. Myocilin stimulates the formation of focal contacts and actin stress fibres in podocytes and other cell types, effects that are mediated through the Wnt signalling pathway. Here, we tested if the expression of both proteins is modified during puromycin aminonucleoside (PAN) nephrosis, which leads to structural changes in the actin cytoskeleton of podocytes. Methods. Rats were treated with PAN, and the effectiveness of treatment was analysed by electron microscopy of podocytes and protein detection in the urine. The expression of Olfm-1 and Myocilin was studied by immunohistochemistry, western blot analysis of glomerular proteins and real-time RT–PCR of glomerular proteins. In parallel experiments, the expression of Olfm-1 was studied in cultured podocytes treated with dexamethasone, TGF-β, TNF-α and PAN. Results. Between Days 5 and 22 after treatment, the amounts of the BMZ and BMY splice variants of Olfm-1 and their mRNA were markedly elevated in proteins and mRNA from isolated glomeruli. Immunohistochemistry showed that the expression of Olfm-1 was confined to podocytes. Essentially, comparable results were obtained for Myocilin. The BMZ variant of Olfm-1 appeared to be secreted from podocytes and was found in high amounts in urine of treated animals. Treatment of cultured podocytes with dexamethasone and PAN caused an increase in Olfm-1 expression, while treatment with recombinant Olfm-1 increased the formation of actin stress fibres. Conclusions. Olfm-1 and Myocilin are markedly induced in podocytes during PAN nephrosis and appear to be involved in the processes that govern the reorganization of the actin cytoskeleton during podocyte repair.
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elevated amounts of Myocilin in the aqueous humor of transgenic mice cause significant changes in ocular gene expression
Experimental Eye Research, 2008Co-Authors: Walter Paper, Paul Russell, Markus Kroeber, Sebastian Heersink, Dietrich A Stephan, Rudolf Fuchshofer, Ernst R. TammAbstract:Myocilin is a 55-57kDa secreted glycoprotein and member of the olfactomedin family, which is mutated in some forms of primary open-angle glaucoma. To assess the effects of elevated amounts of Myocilin on aqueous humor outflow dynamics in an in vivo system, transgenic betaB1-crystallin-MYOC mice have been developed that strongly overexpress Myocilin in their eyes. The transgenic overexpression of Myocilin results in an almost five-fold increase of secreted normal Myocilin in the aqueous humor of betaB1-crystallin-MYOC mice. In the present study, we wanted to use betaB1-crystallin-MYOC as a tool to identify the response of ocular tissues to the presence of higher than normal amounts of Myocilin, and to identify changes in gene expression that could help to shed light on the functional in vivo properties of Myocilin. RNA was isolated from ocular tissues of betaB1-crystallin-MYOC mice and wild-type littermates. Changes in gene expression were determined by hybridization of gene microarrays and confirmed by real time RT-PCR and Western blotting. The expression of genes that had been found to be differentially regulated in betaB1-crystallin-MYOC mice was further analyzed in cultured human trabecular meshwork (HTM) cells treated with recombinant Myocilin. Although betaB1-crystallin-MYOC mice do not have an obvious phenotype, a statistically significant up- and downregulation of several distinct genes was found when compared to gene expression in wild-type littermates. Among the genes that were found to be differentially regulated were Wasl, Ceacam1, and Spon2, which are involved in cell adhesion and cell-matrix interactions. Differences in expression were also found for Six1 which encodes for a transcription factor, and for Pftk1 whose gene product is a cdc2-related protein kinase. The expression of these genes was also found to be regulated in vitro in HTM cells treated with recombinant Myocilin. Substantially higher amounts in ocular tissues of betaB1-crystallin-MYOC mice were found for connexin 46 and alphaB-crystallin. In addition, several genes that encode for olfactomedin proteins showed distinct changes in expression. Olfml3 was significantly downregulated, while Lphn1, Lphn2, and Lphn3 were significantly upregulated. Our findings support a role for Myocilin in modulating cellular adhesion, and suggest functional processes that involve other proteins of the olfactomedin family.
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Myocilin is expressed in the glomerulus of the kidney and induced in mesangioproliferative glomerulonephritis
Kidney International, 2005Co-Authors: Andreas Goldwich, Daniela C Baulmann, Andreas Ohlmann, Cassandra Flugelkoch, Harald O Schocklmann, Ernst R. TammAbstract:Myocilin is expressed in the glomerulus of the kidney and induced in mesangioproliferative glomerulonephritis. Background Myocilin is a 55 to 57 kD secreted glycoprotein and member of the olfactomedin protein family. It is expressed in high amounts in the outflow tissues of the aqueous humor in the eye where it is supposed to contribute to outflow resistance. Myocilin is mutated in some forms of primary open angle glaucoma and affected patients show very high intraocular pressures because of an increase in resistance to aqueous humor outflow. To obtain information, if Myocilin may play a comparable role in other tissues with transendothelial fluid flow, we investigated its expression in the rat kidney. Methods The expression of Myocilin in the normal rat kidney and its changes during mesangioproliferative glomerulonephritis were investigated by immunohistochemistry, one- and two-dimensional gel electrophoresis with Western blotting, and reverse transcription-polymerase chain reaction (RT-PCR). Results Myocilin and its mRNA were detected in isolated glomeruli. Immunohistochemistry showed specific labeling of glomerular cells, while tubular and interstitial regions were essentially negative. Double staining with the podocyte-specific markers synaptopodin and ezrin indicated that Myocilin-positive cells were predominately podocytes. During mesangioproliferative glomerulonephritis, an induction of Myocilin immunoreactivity was observed. Labeling for Myocilin was now observed in activated mesangial cells and areas of glomerular sclerosis. In parallel cell culture experiments, mRNA for Myocilin was detected in cultured murine podocytes and rat mesangial cells. Conclusion Myocilin is expressed in podocytes of the kidney and induced in mesangial cells during experimental mesangioproliferative glomerulonephritis. The specific function of Myocilin in the kidney is not clear, but in a parallel to functions of other olfactomedin proteins, it might have a role in cell-cell adhesion and/or signaling processes.
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overexpression and properties of wild type and tyr437his mutated Myocilin in the eyes of transgenic mice
Investigative Ophthalmology & Visual Science, 2005Co-Authors: Markus Zillig, Paul Russell, Antje Wurm, Franz Grehn, Ernst R. TammAbstract:PURPOSE To obtain experimental in vivo information on the functional properties of Myocilin for aqueous humor (AH) outflow and to study in vivo the processing of mutated Tyr437His Myocilin. Myocilin is a secreted glycoprotein that is mutated in some forms of primary open-angle glaucoma (POAG), and patients with the Tyr437His mutation have severe phenotypes. METHODS The chicken betaB1-crystallin promoter was used to overexpress wild-type human Myocilin and mutated Tyr437His Myocilin in the lenses of transgenic mice. Expression of transgenic mRNA was monitored by Northern blot analysis and in situ hybridization. The localization and secretion of transgenic Myocilin was investigated by Western blot analysis and light and electron microscopy. Intraocular pressure (IOP) was measured by anterior chamber cannulation. RESULTS Two independent lines were established with each of the constructs that showed a strong expression of transgenic mRNA in their lenses. Transgenic expression resulted in a 4.7 +/- 1.8-fold increase of secreted normal Myocilin in mouse AH, compared with its concentration in human AH. Immunoreactivity for transgenic Myocilin was observed along the surfaces of lens and corneal endothelium, and in the chamber angle. At 12 weeks of age, the ultrastructure of the trabecular meshwork in mice expressing normal Myocilin was not different from that of control eyes, and IOP of transgenic animals did not significantly differ from that of control littermates. In contrast, mutated Tyr437His Myocilin was not secreted from lens fibers, but accumulated in dilated cisterns of rough endoplasmic reticulum. Although no structural changes were observed in lenses of animals expressing normal Myocilin, lenses with Tyr437His expression developed nuclear cataracts, completely lost transparency, and eventually ruptured. Structural changes in lenses of Tyr437His expressing mice were correlated with a significant increase in IOP. CONCLUSIONS The results do not support the concept that increasing amounts of Myocilin in the outflow tissues obstruct the system and directly cause an increase in outflow resistance. Mutated Tyr437His Myocilin is not secreted in vivo and causes severe alterations of cellular structure and function. A comparable mechanism may cause POAG in patients with Myocilin mutations.
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perfusion with the olfactomedin domain of Myocilin does not affect outflow facility
Investigative Ophthalmology & Visual Science, 2003Co-Authors: Andreas Goldwich, Ross C Ethier, Darren W H Chan, Ernst R. TammAbstract:Purpose Mutations in the MYOC gene coding for Myocilin are associated with elevated intraocular pressure (IOP), and recombinant Myocilin, produced in a prokaryotic expression system, has been reported to affect aqueous outflow facility. This study was conducted to test whether perfusion with a fragment of recombinant Myocilin (containing the full-length olfactomedin domain), produced in a eukaryotic expression system, affects facility. Methods 293 EBNA cells were transfected by a vector containing the BM40 signal peptide, a human cDNA coding for Myocilin, and a polyhistidine tag (HisTag) sequence. Recombinant protein was isolated by Ni-chelate chromatography, and characterized, and perfused into cultured anterior segments of human and porcine eyes. Results Recombinant Myocilin was secreted as a approximately 55-kDa intact protein and two fragments arising from cleavage of the recombinant protein at amino acid 215. The C-terminal fragment, containing the entire olfactomedin domain, was successfully isolated. When perfused into human and porcine eyes, this C-terminal fragment did not appreciably affect outflow facility. Conclusions Although the olfactomedin domain appears to be important for the function of Myocilin, perfusion with a recombinant Myocilin fragment containing this domain does not change outflow facility. It is possible that both the olfactomedin and N-terminal domains (including the leucine zipper) must be present for Myocilin to have full function. Alternatively, posttranslational modifications of Myocilin may have a major impact on protein function.
Julio Escribano - One of the best experts on this subject based on the ideXlab platform.
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Identification of Myocilin as a blood plasma protein and analysis of its role in leukocyte adhesion to endothelial cell monolayers
2018Co-Authors: José-daniel Aroca-aguilar, Ana Fernández-navarro, Jesús Ontañón, Miguel Coca-prados, Julio EscribanoAbstract:Myocilin is an extracellular glycoprotein with a poorly understood biological function and typically known because of its association with glaucoma. In this study, we analyzed the expression and biological activity of human Myocilin in some non-ocular tissues. Western immunoblot showed the presence of Myocilin in blood plasma as well as in liver and lymphoid tissues (thymus and lymph node). Quantitative PCR confirmed the expression of MYOC in these lymphoid organs and revealed that its mRNA is also present in T-lymphocytes and leukocytes. In addition, detection of 30 kDa C-terminal Myocilin fragments in thymus and liver suggested that Myocilin undergoes an in vivo proteolytic processing that might regulate its biological activity. The presence of Myocilin in blood was further corroborated by peptide mass fingerprinting of the HPLC-isolated protein, and gross estimation of its concentration by Western immunoblot indicated that it is a medium-abundance serum protein with an approximate concentration of 0.85 mg/ml (15.5 μM). Finally, in vitro analyses indicated that Myocilin acts as an anti-adhesive protein for human circulating leukocytes incubated with endothelial cell monolayers. Altogether, these data provide insightful information on new biological properties of Myocilin and suggest its putative role as a blood matricellular protein.
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Immunodetection of Myocilin in human blood serum and plasma by Western blotting.
2018Co-Authors: José-daniel Aroca-aguilar, Ana Fernández-navarro, Jesús Ontañón, Miguel Coca-prados, Julio EscribanoAbstract:Samples of human serum and plasma (30 μg total protein each), and albumin and IgG depleted human plasma (40 μg total protein) were included in the analysis. Positive controls consisted of culture medium containing recombinant Myocilin (rMyoc) expressed in HEK-293T cells and one microgram of HPLC-purified recombinant Myocilin (prMyoc). As a control of depletion, a sample of culture medium containing recombinant Myocilin was treated in parallel (Depl. rMyoc). Electrophoresis was performed on an 8% polyacrylamide gel and the anti-Myocilin C21A polyclonal antibody was used. As a negative immunodetection control, samples were analyzed in parallel with the corresponding preimmune antibody (Preimmune). Exposure time: 1 min. The asterisk indicates the position of non-specific albumin signals. The white arrowhead indicates the position of a Myocilin aggregate. The black arrowhead shows the altered electrophoretic mobility of Myocilin in serum and plasma produced by albumin and the black arrow indicate the position of the recombinant Myocilin monomer in the different samples. Full-length blots and Ponceau S stained membranes are presented in S4 Fig.
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Estimation of Myocilin concentration in human blood serum by Western immunoblot.
2018Co-Authors: José-daniel Aroca-aguilar, Ana Fernández-navarro, Jesús Ontañón, Miguel Coca-prados, Julio EscribanoAbstract:(A) Human blood serum aliquots (30 μg total protein) from control subjects (1–12) were analyzed by Western immunoblot using the chicken polyclonal anti-Myocilin C21A antibody. Samples containing 0.5 μg and 1 μg of HPLC purified recombinant human Myocilin were used as a reference for densitometry. Each sample was analyzed in triplicate. The image shows a representative Western blot. The full-length membrane is shown in S5 Fig. (B) Estimated Myocilin concentration in blood serum samples from 12 human donors. Values are expressed as mean ± SEM of triplicates from two independent analyses. rMyoc: recombinant Myocilin.
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Analysis of Myocilin expression in human lymphoid tissues and leukocytes by RT-qPCR.
2018Co-Authors: José-daniel Aroca-aguilar, Ana Fernández-navarro, Jesús Ontañón, Miguel Coca-prados, Julio EscribanoAbstract:cDNAs (100 ng) were used to amplify Myocilin mRNA as indicated in Materials and Methods. The amount of mRNA detected in lymph node was used as a reference to calculate the fold-change of Myocilin mRNA. Values are expressed as mean ± SEM of at least three independent experiments carried out in triplicate. Asterisks indicate statistical significance as compared to lymph node: p
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Analysis of Myocilin expression in human tissues by Western immunoblot.
2018Co-Authors: José-daniel Aroca-aguilar, Ana Fernández-navarro, Jesús Ontañón, Miguel Coca-prados, Julio EscribanoAbstract:An aliquot of human plasma and lysates of the different tissues (20 μg total protein), were analyzed by SDS-PAGE. HPLC purified recombinant human Myocilin (0.5 μg) was used as a positive control (prMyoc). Myocilin was detected by Western immunoblotting using a chicken IgY polyclonal antibody (C21A). As a negative control a replica of the membrane was analyzed in parallel with the preimmune antibody. Exposure time: 30 s. The Ponceau stained membranes are presented in S2 Fig.
Beatrice Y. J. T. Yue - One of the best experts on this subject based on the ideXlab platform.
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cellular processing of Myocilin
PLOS ONE, 2014Co-Authors: Ye Qiu, Xiang Shen, Rajalekshmy Shyam, Beatrice Y. J. T. Yue, Hongyu YingAbstract:Background Myocilin (MYOC) is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Pro370Leu (P370L) and Gln368stop (Q368X) have been identified in patients. In the present study, we investigated the processing of Myocilin in human trabecular meshwork (TM) cells as well as in inducible, stable RGC5 cell lines.
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Effects of inhibitors on levels of endogenous Myocilin.
2014Co-Authors: Ye Qiu, Xiang Shen, Rajalekshmy Shyam, Beatrice Y. J. T. Yue, Hongyu YingAbstract:A. Western blotting. Human TM cells were treated for 16 h with vehicle DMSO or H2O (not shown), or proteasomal (LCT and epoxomicin), lysosomal (NH4Cl and chloroquin) or autophagic (3-MA) inhibitors. Proteins (25 µg) in cell lysates were immunoblotted with anti-Myocilin or anti-GAPDH. Densitometry was performed. The Myocilin/GAPDH relative to the DMSO control ratios are presented. B. Equal aliquots of media were immunoblotted with anti-Myocilin. The level of Myocilin in the media was normalized to that of the DMSO control. C. Immunofluorescence for Myocilin. TM cells treated as above with the various inhibitors were fixed and immunostained for Myocilin. All experiments were repeated at least 3 times, yielding similar results. Scale bar, 20 µm.
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Effects of inhibitors on levels of expressed Myocilin-GFP in inducible cells.
2014Co-Authors: Ye Qiu, Xiang Shen, Rajalekshmy Shyam, Beatrice Y. J. T. Yue, Hongyu YingAbstract:Fluorescence images of MyocilinWT-GFP (MyocWT-GFP, A), MyocilinP370L-GFP (MyocP370L-GFP, C), MyocilinQ368X-GFP (MyocQ368X-GFP, E)-expressing-RGC5 cells treated with various inhibitors are shown. Scale bar, 20 µm. For Western blotting, RGC5 cells induced by Dox for 24 h to express MyocilinWT-GFP (B), MyocilinP370L-GFP (D), and MyocilinQ368X-GFP (F) were treated for 16 h with vehicle DMSO, or proteasomal (LCT and epoxomicin), lysosomal (NH4Cl and chloroquin) or autophagic (3-MA) inhibitors. Proteins (25 µg) in cell lysates were immunoblotted with anti-Myocilin or anti-GAPDH. Protein bands for the endogenous Myocilin (55/57 kDa) and Myocilin-GFP (∼100 kDa) were seen. Densitometry was performed. The Myocilin-GFP/GAPDH relative to the DMSO control ratios are presented. All experiments were repeated at least 3 times.
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differential effects of Myocilin and optineurin two glaucoma genes on neurite outgrowth
American Journal of Pathology, 2010Co-Authors: Takahisa Koga, Bum-chan Park, Xiang Shen, Ye Qiu, Rajalekshmy Shyam, Jeongseok Park, Beatrice Y. J. T. YueAbstract:Myocilin and optineurin are two genes linked to glaucoma, a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. To investigate the effects of force-expressed wild-type and mutant Myocilin and optineurin on neurite outgrowth in neuronal cells, we transiently transfected cells with pEGFP-N1 (mock control) as well as Myocilin and optineurin plasmids including pMYOCWT-EGFP, pMYOCP370L-EGFP, pMYOC1-367-EGFP, pOPTNWT-EGFP, and pOPTNE50K-EGFP. PC12 cells transfected with pEGFP-N1 produced, as anticipated, long and extensive neuritis on nerve growth factor induction. The neurite length in those cells transfected with Myocilin constructs was shortened and the number of neurites was also reduced. A similar inhibitory effect on neurite outgrowth was also elicited by Myocilin transfection in RGC5 cells. In contrast, neither transfection of the optineurin constructs pOPTNWT-EGFP and pOPTNE50K-EGFP nor the Myocilin and optineurin small-interfering RNA treatments induced significant alterations in neurite outgrowth. Transfection with the wild-type optineurin construct, but not with that of the wild-type Myocilin, increased the apoptotic activity in cells. These results demonstrated that the two glaucoma genes, Myocilin and optineurin, exhibited differential effects on neurite outgrowth. They may contribute to the development of neurodegenerative glaucoma via distinct mechanisms.
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interaction of Myocilin with gamma synuclein affects its secretion and aggregation
Cellular and Molecular Neurobiology, 2005Co-Authors: Irina Surgucheva, Stanislav I. Tomarev, Bum-chan Park, Beatrice Y. J. T. Yue, Andrei SurguchovAbstract:Mutations in the gene encoding human Myocilin are associated with some cases of juvenile and early-onset glaucoma. Glaucomatous mutations prevent Myocilin from being secreted. The analysis of the defects associated with mutations point to the existence of factor(s) in addition to mutations that might be implicated in the development of glaucoma. In the present paper, we found that interaction of Myocilin with one of the members of the synuclein family alters its properties, including its ability to be secreted. Results of immunoprecipitation show that Myocilin is a gamma-synuclein-interacting protein. Further analysis demonstrated that both Myocilin and gamma-synuclein are expressed in human TM cells, immortalized rat ganglion (RGC-5) cells, and HT22 hippocampal neurons. According to Western blotting, in addition to monomeric form with molecular weight 17 kDa gamma-synuclein is present as higher molecular weight forms ( approximately 35 and 68 KDa), presumably dimer and tetramer. Myocilin and gamma-synuclein have partially overlapping perinuclear localization. Dexamethasone upregulates Myocilin expression in RGC-5 cells and HT22 hippocampal neurons. We found alterations of Myocilin properties as a result of its interaction with gamma-synuclein. In cultured cells, gamma-synuclein upregulates Myocilin expression, inhibits its secretion and prevents the formation of high molecular weight forms of Myocilin. Although both alpha-synuclein and gamma-synuclein are expressed in HTM cells, only gamma-synuclein interacts with Myocilin and alters its properties. We conclude that Myocilin and gamma-synuclein interact and as a result, Myocilin's properties are changed. Since Myocilin and gamma-synuclein have partially overlapping intracellular localization in cell types that are implicated in glaucoma development, their interaction may play an important role in glaucoma.
